Difference between revisions of "Team:Macquarie Australia/Experiments"
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech</a></li> | <li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech</a></li> | ||
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+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:#666666; | ||
+ | font-weight:normal; | ||
+ | font-style:italic;} | ||
+ | h6.CxSpLast | ||
+ | {mso-style-link:"Heading 6 Char"; | ||
+ | mso-style-next:"Normal\,normal"; | ||
+ | mso-style-type:export-only; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-add-space:auto; | ||
+ | line-height:115%; | ||
+ | mso-pagination:widow-orphan lines-together; | ||
+ | page-break-after:avoid; | ||
+ | mso-outline-level:6; | ||
+ | font-size:11.0pt; | ||
+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:#666666; | ||
+ | font-weight:normal; | ||
+ | font-style:italic;} | ||
+ | p.MsoTitle, li.MsoTitle, div.MsoTitle | ||
+ | {mso-style-link:"Title Char"; | ||
+ | mso-style-next:"Normal\,normal"; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-add-space:auto; | ||
+ | line-height:115%; | ||
+ | mso-pagination:widow-orphan lines-together; | ||
+ | page-break-after:avoid; | ||
+ | font-size:21.0pt; | ||
+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:black;} | ||
+ | p.MsoTitleCxSpFirst, li.MsoTitleCxSpFirst, div.MsoTitleCxSpFirst | ||
+ | {mso-style-link:"Title Char"; | ||
+ | mso-style-next:"Normal\,normal"; | ||
+ | mso-style-type:export-only; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-add-space:auto; | ||
+ | line-height:115%; | ||
+ | mso-pagination:widow-orphan lines-together; | ||
+ | page-break-after:avoid; | ||
+ | font-size:21.0pt; | ||
+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:black;} | ||
+ | p.MsoTitleCxSpMiddle, li.MsoTitleCxSpMiddle, div.MsoTitleCxSpMiddle | ||
+ | {mso-style-link:"Title Char"; | ||
+ | mso-style-next:"Normal\,normal"; | ||
+ | mso-style-type:export-only; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-add-space:auto; | ||
+ | line-height:115%; | ||
+ | mso-pagination:widow-orphan lines-together; | ||
+ | page-break-after:avoid; | ||
+ | font-size:21.0pt; | ||
+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:black;} | ||
+ | p.MsoTitleCxSpLast, li.MsoTitleCxSpLast, div.MsoTitleCxSpLast | ||
+ | {mso-style-link:"Title Char"; | ||
+ | mso-style-next:"Normal\,normal"; | ||
+ | mso-style-type:export-only; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-add-space:auto; | ||
+ | line-height:115%; | ||
+ | mso-pagination:widow-orphan lines-together; | ||
+ | page-break-after:avoid; | ||
+ | font-size:21.0pt; | ||
+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:black;} | ||
+ | p.MsoSubtitle, li.MsoSubtitle, div.MsoSubtitle | ||
+ | {mso-style-link:"Subtitle Char"; | ||
+ | mso-style-next:"Normal\,normal"; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-add-space:auto; | ||
+ | line-height:115%; | ||
+ | mso-pagination:widow-orphan lines-together; | ||
+ | page-break-after:avoid; | ||
+ | font-size:13.0pt; | ||
+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:#666666; | ||
+ | font-style:italic;} | ||
+ | p.MsoSubtitleCxSpFirst, li.MsoSubtitleCxSpFirst, div.MsoSubtitleCxSpFirst | ||
+ | {mso-style-link:"Subtitle Char"; | ||
+ | mso-style-next:"Normal\,normal"; | ||
+ | mso-style-type:export-only; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-add-space:auto; | ||
+ | line-height:115%; | ||
+ | mso-pagination:widow-orphan lines-together; | ||
+ | page-break-after:avoid; | ||
+ | font-size:13.0pt; | ||
+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:#666666; | ||
+ | font-style:italic;} | ||
+ | p.MsoSubtitleCxSpMiddle, li.MsoSubtitleCxSpMiddle, div.MsoSubtitleCxSpMiddle | ||
+ | {mso-style-link:"Subtitle Char"; | ||
+ | mso-style-next:"Normal\,normal"; | ||
+ | mso-style-type:export-only; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-add-space:auto; | ||
+ | line-height:115%; | ||
+ | mso-pagination:widow-orphan lines-together; | ||
+ | page-break-after:avoid; | ||
+ | font-size:13.0pt; | ||
+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:#666666; | ||
+ | font-style:italic;} | ||
+ | p.MsoSubtitleCxSpLast, li.MsoSubtitleCxSpLast, div.MsoSubtitleCxSpLast | ||
+ | {mso-style-link:"Subtitle Char"; | ||
+ | mso-style-next:"Normal\,normal"; | ||
+ | mso-style-type:export-only; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-add-space:auto; | ||
+ | line-height:115%; | ||
+ | mso-pagination:widow-orphan lines-together; | ||
+ | page-break-after:avoid; | ||
+ | font-size:13.0pt; | ||
+ | font-family:"Trebuchet MS"; | ||
+ | mso-fareast-font-family:"Trebuchet MS"; | ||
+ | mso-bidi-font-family:"Trebuchet MS"; | ||
+ | color:#666666; | ||
+ | font-style:italic;} | ||
+ | span.Heading1Char | ||
+ | {mso-style-name:"Heading 1 Char"; | ||
+ | mso-style-locked:yes; | ||
+ | mso-style-link:"Heading 1"; | ||
+ | mso-ansi-font-size:16.0pt; | ||
+ | mso-bidi-font-size:16.0pt; | ||
+ | font-family:Calibri; | ||
+ | mso-ascii-font-family:Calibri; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Calibri; | ||
+ | mso-bidi-font-family:"Times New Roman"; | ||
+ | color:#345A8A; | ||
+ | mso-ansi-language:EN-AU; | ||
+ | font-weight:bold;} | ||
+ | span.Heading2Char | ||
+ | {mso-style-name:"Heading 2 Char"; | ||
+ | mso-style-noshow:yes; | ||
+ | mso-style-locked:yes; | ||
+ | mso-style-link:"Heading 2"; | ||
+ | mso-ansi-font-size:13.0pt; | ||
+ | mso-bidi-font-size:13.0pt; | ||
+ | font-family:Calibri; | ||
+ | mso-ascii-font-family:Calibri; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Calibri; | ||
+ | mso-bidi-font-family:"Times New Roman"; | ||
+ | color:#4F81BD; | ||
+ | mso-ansi-language:EN-AU; | ||
+ | font-weight:bold;} | ||
+ | span.Heading3Char | ||
+ | {mso-style-name:"Heading 3 Char"; | ||
+ | mso-style-noshow:yes; | ||
+ | mso-style-locked:yes; | ||
+ | mso-style-link:"Heading 3"; | ||
+ | mso-ansi-font-size:11.0pt; | ||
+ | mso-bidi-font-size:11.0pt; | ||
+ | font-family:Calibri; | ||
+ | mso-ascii-font-family:Calibri; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Calibri; | ||
+ | mso-bidi-font-family:"Times New Roman"; | ||
+ | color:#4F81BD; | ||
+ | mso-ansi-language:EN-AU; | ||
+ | font-weight:bold;} | ||
+ | span.Heading4Char | ||
+ | {mso-style-name:"Heading 4 Char"; | ||
+ | mso-style-noshow:yes; | ||
+ | mso-style-locked:yes; | ||
+ | mso-style-link:"Heading 4"; | ||
+ | mso-ansi-font-size:11.0pt; | ||
+ | mso-bidi-font-size:11.0pt; | ||
+ | font-family:Calibri; | ||
+ | mso-ascii-font-family:Calibri; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Calibri; | ||
+ | mso-bidi-font-family:"Times New Roman"; | ||
+ | color:#4F81BD; | ||
+ | mso-ansi-language:EN-AU; | ||
+ | font-weight:bold; | ||
+ | font-style:italic;} | ||
+ | span.Heading5Char | ||
+ | {mso-style-name:"Heading 5 Char"; | ||
+ | mso-style-noshow:yes; | ||
+ | mso-style-locked:yes; | ||
+ | mso-style-link:"Heading 5"; | ||
+ | mso-ansi-font-size:11.0pt; | ||
+ | mso-bidi-font-size:11.0pt; | ||
+ | font-family:Calibri; | ||
+ | mso-ascii-font-family:Calibri; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Calibri; | ||
+ | mso-bidi-font-family:"Times New Roman"; | ||
+ | color:#244061; | ||
+ | mso-ansi-language:EN-AU;} | ||
+ | span.Heading6Char | ||
+ | {mso-style-name:"Heading 6 Char"; | ||
+ | mso-style-noshow:yes; | ||
+ | mso-style-locked:yes; | ||
+ | mso-style-link:"Heading 6"; | ||
+ | mso-ansi-font-size:11.0pt; | ||
+ | mso-bidi-font-size:11.0pt; | ||
+ | font-family:Calibri; | ||
+ | mso-ascii-font-family:Calibri; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Calibri; | ||
+ | mso-bidi-font-family:"Times New Roman"; | ||
+ | color:#244061; | ||
+ | mso-ansi-language:EN-AU; | ||
+ | font-style:italic;} | ||
+ | span.TitleChar | ||
+ | {mso-style-name:"Title Char"; | ||
+ | mso-style-locked:yes; | ||
+ | mso-style-link:Title; | ||
+ | mso-ansi-font-size:26.0pt; | ||
+ | mso-bidi-font-size:26.0pt; | ||
+ | font-family:Calibri; | ||
+ | mso-ascii-font-family:Calibri; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Calibri; | ||
+ | mso-bidi-font-family:"Times New Roman"; | ||
+ | color:#183A63; | ||
+ | letter-spacing:.25pt; | ||
+ | mso-font-kerning:14.0pt; | ||
+ | mso-ansi-language:EN-AU;} | ||
+ | span.SubtitleChar | ||
+ | {mso-style-name:"Subtitle Char"; | ||
+ | mso-style-locked:yes; | ||
+ | mso-style-link:Subtitle; | ||
+ | font-family:Calibri; | ||
+ | mso-ascii-font-family:Calibri; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Calibri; | ||
+ | mso-bidi-font-family:"Times New Roman"; | ||
+ | color:#4F81BD; | ||
+ | letter-spacing:.75pt; | ||
+ | mso-ansi-language:EN-AU; | ||
+ | font-style:italic;} | ||
+ | p.normalcxspmiddle, li.normalcxspmiddle, div.normalcxspmiddle | ||
+ | {mso-style-name:normalcxspmiddle; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-pagination:widow-orphan; | ||
+ | font-size:10.0pt; | ||
+ | font-family:"Times New Roman"; | ||
+ | mso-ascii-font-family:Times; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Times; | ||
+ | mso-bidi-font-family:"Times New Roman";} | ||
+ | p.normalcxsplast, li.normalcxsplast, div.normalcxsplast | ||
+ | {mso-style-name:normalcxsplast; | ||
+ | margin:0cm; | ||
+ | margin-bottom:.0001pt; | ||
+ | mso-pagination:widow-orphan; | ||
+ | font-size:10.0pt; | ||
+ | font-family:"Times New Roman"; | ||
+ | mso-ascii-font-family:Times; | ||
+ | mso-fareast-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:Times; | ||
+ | mso-bidi-font-family:"Times New Roman";} | ||
+ | span.SpellE | ||
+ | {mso-style-name:""; | ||
+ | mso-spl-e:yes;} | ||
+ | span.GramE | ||
+ | {mso-style-name:""; | ||
+ | mso-gram-e:yes;} | ||
+ | @page Section1 | ||
+ | {size:612.0pt 792.0pt; | ||
+ | margin:72.0pt 72.0pt 72.0pt 72.0pt; | ||
+ | mso-header-margin:36.0pt; | ||
+ | mso-footer-margin:36.0pt; | ||
+ | mso-page-numbers:1; | ||
+ | mso-paper-source:0;} | ||
+ | div.Section1 | ||
+ | {page:Section1;} | ||
+ | /* List Definitions */ | ||
+ | @list l0 | ||
+ | {mso-list-id:1977220; | ||
+ | mso-list-template-ids:733219686;} | ||
+ | @list l0:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l0:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l0:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l0:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l0:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l0:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l0:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l0:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l0:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l1 | ||
+ | {mso-list-id:53243609; | ||
+ | mso-list-template-ids:-1377519374;} | ||
+ | @list l1:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l1:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l1:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l1:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l1:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l1:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l1:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l1:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l1:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l2 | ||
+ | {mso-list-id:119346809; | ||
+ | mso-list-template-ids:-1607330878;} | ||
+ | @list l2:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l2:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l2:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l2:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l2:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l2:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l2:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l2:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l2:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l3 | ||
+ | {mso-list-id:129523532; | ||
+ | mso-list-template-ids:1017046104;} | ||
+ | @list l3:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l3:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l3:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l3:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l3:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l3:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l3:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l3:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l3:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l4 | ||
+ | {mso-list-id:268048739; | ||
+ | mso-list-template-ids:1087815984;} | ||
+ | @list l4:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l4:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l4:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l4:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l4:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l4:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l4:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l4:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l4:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l5 | ||
+ | {mso-list-id:308486205; | ||
+ | mso-list-template-ids:280934600;} | ||
+ | @list l5:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l5:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l5:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l5:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l5:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l5:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l5:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l5:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l5:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l6 | ||
+ | {mso-list-id:416901852; | ||
+ | mso-list-template-ids:-1999089562;} | ||
+ | @list l6:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l6:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l6:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l6:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l6:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l6:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l6:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l6:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l6:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l7 | ||
+ | {mso-list-id:430708754; | ||
+ | mso-list-template-ids:1530537842;} | ||
+ | @list l7:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l7:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l7:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l7:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l7:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l7:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l7:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l7:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l7:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l8 | ||
+ | {mso-list-id:437674611; | ||
+ | mso-list-template-ids:1467396388;} | ||
+ | @list l8:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l8:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l8:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l8:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l8:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l8:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l8:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l8:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l8:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l9 | ||
+ | {mso-list-id:450592413; | ||
+ | mso-list-template-ids:1449056272;} | ||
+ | @list l9:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l9:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l9:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l9:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l9:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l9:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l9:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l9:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l9:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l10 | ||
+ | {mso-list-id:587423861; | ||
+ | mso-list-template-ids:2021664494;} | ||
+ | @list l10:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l10:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l10:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l10:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l10:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l10:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l10:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l10:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l10:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l11 | ||
+ | {mso-list-id:778992503; | ||
+ | mso-list-template-ids:-1337819496;} | ||
+ | @list l11:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l11:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l11:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l11:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l11:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l11:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l11:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l11:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l11:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l12 | ||
+ | {mso-list-id:782724505; | ||
+ | mso-list-template-ids:-1329422302;} | ||
+ | @list l12:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l12:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l12:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l12:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l12:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l12:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l12:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l12:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l12:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l13 | ||
+ | {mso-list-id:1175072486; | ||
+ | mso-list-template-ids:-590846074;} | ||
+ | @list l13:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l13:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l13:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l13:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l13:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l13:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l13:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l13:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l13:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l14 | ||
+ | {mso-list-id:1291085181; | ||
+ | mso-list-template-ids:-1825172428;} | ||
+ | @list l14:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l14:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l14:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l14:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l14:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l14:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l14:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l14:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l14:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l15 | ||
+ | {mso-list-id:1358972088; | ||
+ | mso-list-template-ids:-21460484;} | ||
+ | @list l15:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l15:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l15:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l15:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l15:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l15:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l15:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l15:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l15:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l16 | ||
+ | {mso-list-id:1363091701; | ||
+ | mso-list-type:hybrid; | ||
+ | mso-list-template-ids:1425705876 67698703 67698713 67698715 67698703 67698713 67698715 67698703 67698713 67698715;} | ||
+ | @list l16:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | margin-left:49.65pt; | ||
+ | text-indent:-18.0pt;} | ||
+ | @list l16:level2 | ||
+ | {mso-level-tab-stop:72.0pt; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:-18.0pt;} | ||
+ | @list l16:level3 | ||
+ | {mso-level-tab-stop:108.0pt; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:-18.0pt;} | ||
+ | @list l16:level4 | ||
+ | {mso-level-tab-stop:144.0pt; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:-18.0pt;} | ||
+ | @list l16:level5 | ||
+ | {mso-level-tab-stop:180.0pt; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:-18.0pt;} | ||
+ | @list l16:level6 | ||
+ | {mso-level-tab-stop:216.0pt; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:-18.0pt;} | ||
+ | @list l16:level7 | ||
+ | {mso-level-tab-stop:252.0pt; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:-18.0pt;} | ||
+ | @list l16:level8 | ||
+ | {mso-level-tab-stop:288.0pt; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:-18.0pt;} | ||
+ | @list l16:level9 | ||
+ | {mso-level-tab-stop:324.0pt; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:-18.0pt;} | ||
+ | @list l17 | ||
+ | {mso-list-id:1390767940; | ||
+ | mso-list-template-ids:-916832514;} | ||
+ | @list l17:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l17:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l17:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l17:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l17:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l17:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l17:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l17:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l17:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l18 | ||
+ | {mso-list-id:1391926166; | ||
+ | mso-list-template-ids:305986318;} | ||
+ | @list l18:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l18:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l18:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l18:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l18:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l18:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l18:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l18:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l18:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l19 | ||
+ | {mso-list-id:1442073371; | ||
+ | mso-list-template-ids:410680830;} | ||
+ | @list l19:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l19:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l19:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l19:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l19:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l19:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l19:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l19:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l19:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l20 | ||
+ | {mso-list-id:1572617164; | ||
+ | mso-list-template-ids:-1956855408;} | ||
+ | @list l20:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l20:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l20:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l20:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l20:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l20:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l20:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l20:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l20:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l21 | ||
+ | {mso-list-id:1580409131; | ||
+ | mso-list-template-ids:1448512234;} | ||
+ | @list l21:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l21:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l21:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l21:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l21:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l21:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l21:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l21:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l21:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l22 | ||
+ | {mso-list-id:1596789075; | ||
+ | mso-list-template-ids:2116564854;} | ||
+ | @list l22:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l22:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l22:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l22:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l22:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l22:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l22:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l22:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l22:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l23 | ||
+ | {mso-list-id:1658459251; | ||
+ | mso-list-template-ids:1389007528;} | ||
+ | @list l23:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l23:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l23:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l23:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l23:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l23:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l23:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l23:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l23:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l24 | ||
+ | {mso-list-id:1690060605; | ||
+ | mso-list-template-ids:1494383072;} | ||
+ | @list l24:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l24:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l24:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l24:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l24:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l24:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l24:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l24:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l24:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l25 | ||
+ | {mso-list-id:1697467125; | ||
+ | mso-list-template-ids:-1286720966;} | ||
+ | @list l25:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l25:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l25:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l25:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l25:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l25:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l25:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l25:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l25:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l26 | ||
+ | {mso-list-id:1819763611; | ||
+ | mso-list-template-ids:353253044;} | ||
+ | @list l26:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l26:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l26:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l26:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l26:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l26:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l26:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l26:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l26:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l27 | ||
+ | {mso-list-id:1881092920; | ||
+ | mso-list-template-ids:851230504;} | ||
+ | @list l27:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l27:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l27:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l27:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l27:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l27:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l27:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l27:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l27:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l28 | ||
+ | {mso-list-id:1890534047; | ||
+ | mso-list-template-ids:625272808;} | ||
+ | @list l28:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l28:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l28:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l28:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l28:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l28:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l28:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l28:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l28:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l29 | ||
+ | {mso-list-id:1923950169; | ||
+ | mso-list-template-ids:-1764827070;} | ||
+ | @list l29:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l29:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l29:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l29:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l29:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l29:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l29:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l29:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l29:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l30 | ||
+ | {mso-list-id:1936936593; | ||
+ | mso-list-template-ids:-1352398384;} | ||
+ | @list l30:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l30:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l30:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l30:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l30:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l30:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l30:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l30:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l30:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l31 | ||
+ | {mso-list-id:1942713080; | ||
+ | mso-list-template-ids:-1074340452;} | ||
+ | @list l31:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l31:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l31:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l31:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l31:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l31:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l31:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l31:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l31:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l32 | ||
+ | {mso-list-id:1946158209; | ||
+ | mso-list-template-ids:-842081000;} | ||
+ | @list l32:level1 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-ansi-font-size:12.0pt; | ||
+ | mso-ascii-font-family:Arial; | ||
+ | mso-fareast-font-family:Arial; | ||
+ | mso-hansi-font-family:Arial; | ||
+ | mso-bidi-font-family:Cambria; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l32:level2 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l32:level3 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l32:level4 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l32:level5 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l32:level6 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:198.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l32:level7 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CF; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:234.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l32:level8 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25CB; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l32:level9 | ||
+ | {mso-level-number-format:bullet; | ||
+ | mso-level-text:\25A0; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:306.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l33 | ||
+ | {mso-list-id:1965041241; | ||
+ | mso-list-template-ids:-1765666866;} | ||
+ | @list l33:level1 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:18.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l33:level2 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:54.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l33:level3 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:90.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l33:level4 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:126.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l33:level5 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:162.0pt; | ||
+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l33:level6 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:198.0pt; | ||
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+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l33:level7 | ||
+ | {mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
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+ | mso-text-animation:none; | ||
+ | text-decoration:none; | ||
+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l33:level8 | ||
+ | {mso-level-number-format:alpha-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:left; | ||
+ | text-indent:270.0pt; | ||
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+ | text-underline:none; | ||
+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | @list l33:level9 | ||
+ | {mso-level-number-format:roman-lower; | ||
+ | mso-level-tab-stop:none; | ||
+ | mso-level-number-position:right; | ||
+ | text-indent:306.0pt; | ||
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+ | text-decoration:none; | ||
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+ | text-decoration:none; | ||
+ | text-line-through:none;} | ||
+ | ol | ||
+ | {margin-bottom:0cm;} | ||
+ | ul | ||
+ | {margin-bottom:0cm;} | ||
+ | --> | ||
+ | </style> | ||
+ | <!--[if gte mso 10]> | ||
+ | <style> | ||
+ | /* Style Definitions */ | ||
+ | table.MsoNormalTable | ||
+ | {mso-style-name:"Table Normal"; | ||
+ | mso-tstyle-rowband-size:0; | ||
+ | mso-tstyle-colband-size:0; | ||
+ | mso-style-noshow:yes; | ||
+ | mso-style-parent:""; | ||
+ | mso-padding-alt:0cm 5.4pt 0cm 5.4pt; | ||
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+ | mso-para-margin-bottom:.0001pt; | ||
+ | mso-pagination:widow-orphan; | ||
+ | font-size:10.0pt; | ||
+ | font-family:"Times New Roman"; | ||
+ | mso-ascii-font-family:Cambria; | ||
+ | mso-hansi-font-family:Cambria;} | ||
+ | table.MsoTableGrid | ||
+ | {mso-style-name:"Table Grid"; | ||
+ | mso-tstyle-rowband-size:0; | ||
+ | mso-tstyle-colband-size:0; | ||
+ | border:solid black; | ||
+ | mso-border-themecolor:text1; | ||
+ | border:1.0pt; | ||
+ | mso-border-alt:solid black; | ||
+ | mso-border-themecolor:text1; | ||
+ | mso-border-alt:.5pt; | ||
+ | mso-padding-alt:0cm 5.4pt 0cm 5.4pt; | ||
+ | mso-border-insideh:.5pt solid black; | ||
+ | mso-border-insideh-themecolor:text1; | ||
+ | mso-border-insidev:.5pt solid black; | ||
+ | mso-border-insidev-themecolor:text1; | ||
+ | mso-para-margin:0cm; | ||
+ | mso-para-margin-bottom:.0001pt; | ||
+ | mso-pagination:widow-orphan; | ||
+ | font-size:10.0pt; | ||
+ | font-family:"Times New Roman"; | ||
+ | mso-ascii-font-family:Cambria; | ||
+ | mso-hansi-font-family:Cambria;} | ||
+ | </style> | ||
+ | <![endif]--><!--[if gte mso 9]><xml> | ||
+ | <o:shapedefaults v:ext="edit" spidmax="2050"/> | ||
+ | </xml><![endif]--><!--[if gte mso 9]><xml> | ||
+ | <o:shapelayout v:ext="edit"> | ||
+ | <o:idmap v:ext="edit" data="1"/> | ||
+ | </o:shapelayout></xml><![endif]--> | ||
+ | </head> | ||
+ | |||
+ | <body bgcolor=white lang=EN-AU style='tab-interval:36.0pt'> | ||
+ | |||
+ | <div class=Section1> | ||
+ | |||
+ | <p class=MsoNormal><u>Protocols </u></p> | ||
+ | |||
+ | <p class=MsoNormal align=center style='text-align:center'><u><span | ||
+ | style='font-size:14.0pt;line-height:115%'>Making competent cells</span></u></p> | ||
+ | |||
+ | <p class=MsoNormal><o:p> </o:p></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:49.65pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Using a sterile | ||
+ | plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. | ||
+ | Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22<sup>o</sup>C, | ||
+ | 200-250rpm.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:14.2pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-.55pt'><span style='font-size:14.0pt'><o:p> </o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:49.65pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>A600 should be | ||
+ | 0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~ | ||
+ | 0.5.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:14.2pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-.55pt'><span style='font-size:14.0pt'><o:p> </o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>3.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Remove the flask from the incubator and place on ice | ||
+ | for 10 minutes.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>4.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Transfer the culture to a 15mL centrifuge tube and | ||
+ | spin at 2500 x g for 10 min at 4<sup>o</sup>C<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>5.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Pour off and discard the supernatant, and immediately | ||
+ | place the tube on ice.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>6.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Resuspend your cells in 1mL of ice-cold TB buffer, | ||
+ | make sure there are no clumps of cells left, but also treat your cells gently | ||
+ | and keep them cold.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>7.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Add ice-cold TB buffer to bring volume up to 1/5th of | ||
+ | the original culture volume (~30mL in this case). Mix the tube by gently | ||
+ | inverting 3 times.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>8.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Incubate the tube on ice for 10 minutes.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>9.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Centrifuge at 2<span class=GramE>,500 x</span> g for 7 | ||
+ | minutes at 4<sup>o</sup>C, discard the supernatant.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>10.<span | ||
+ | style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Gently resuspend | ||
+ | the cells in ~1/20th of the original culture volume of ice-cold TB buffer. | ||
+ | NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. <span | ||
+ | class=GramE>e</span>.g. if the culture OD600 was 0.1 then resuspend in 1/100th | ||
+ | of original volume.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>11.<span | ||
+ | style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Pre-chill 1.5ml <span | ||
+ | class=SpellE>Eppendorf</span> tubes on ice. Add 930µl of your cell suspension, | ||
+ | keeping the remainder on ice in the 15mL tube.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>12.<span | ||
+ | style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Add 70µl of DMSO | ||
+ | to the 930µl of cell suspension. Mix gently by swirling, and place on ice.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>13.<span | ||
+ | style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Aliquot 100µl of | ||
+ | the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the | ||
+ | tubes with: Date – Strain. <span class=GramE>Snap freeze</span> with | ||
+ | liquid nitrogen.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent: | ||
+ | -18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>14.<span | ||
+ | style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Repeat step 11-13 | ||
+ | for the rest of your cell suspension in step 10. Store cells at -80<sup>o</sup>C<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.97a2w31llx4p></a><o:p><span style='text-decoration:none'> </span></o:p></h4> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.33k2zqtujk1j></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333'>TB BUFFER</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l7 level1 lfo2'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span | ||
+ | style='font-size:14.0pt'> 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g <span | ||
+ | class=SpellE>KCl</span>.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l7 level1 lfo2'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span | ||
+ | style='font-size:14.0pt'> All components (except for MnCl2-4H2O) were mixed and | ||
+ | dissolved in 500 <span class=SpellE>mL</span> of water and pH adjusted to 6.7 | ||
+ | with KOH. Then, MnCl2-4H2O, was dissolved in 300 <span class=SpellE>mL</span> | ||
+ | of water, mixed and solution adjusted to 1 L. Sterilisation via filtration | ||
+ | followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.77uif8qp8qn4></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333'>EDTA BUFFER</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l0 level1 lfo3'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span | ||
+ | style='font-size:14.0pt'> 37.22g EDTA solid, 180 <span class=SpellE>mL</span> | ||
+ | of water and pH adjusted to 8.0 using 10M <span class=SpellE>NaOH</span>.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l0 level1 lfo3'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span | ||
+ | style='font-size:14.0pt'> Components were combined then pH adjusted.<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.q84g5o6q56lo></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333'>TAE BUFFER</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l23 level1 lfo4'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span | ||
+ | style='font-size:14.0pt'> 121.2g <span class=SpellE>Tris</span> base (dissolved | ||
+ | in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0).<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l23 level1 lfo4'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span | ||
+ | style='font-size:14.0pt'> A total volume of 500 <span class=SpellE>mL</span> | ||
+ | was made up as a 50x stock solution using all components<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.2oiikqt7my41></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333'>Making agarose gel</span></b></h4> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.ffz7g0pkhkr5></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333'>Preparing the Gel </span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l17 level1 lfo5'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Mix 1g of agarose powder with 100ml of 1x TAE buffer | ||
+ | and heat for 1minute or until all agarose is dissolved.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l17 level1 lfo5'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Wait until it has | ||
+ | cooled (not set), and add 1ul of <span class=SpellE>GelRed</span> into the | ||
+ | mixture. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l17 level1 lfo5'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Pour the solution | ||
+ | into a cast with an appropriate comb.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l17 level1 lfo5'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Leave to set.<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.s2q8apkb2g></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333'>Running the Gel </span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l18 level1 lfo6'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Mix 1ul of 1kbp DNA ladder with 6ul of loading dye (<span | ||
+ | class=SpellE>bromophenol</span> blue) and 4ul of 1x TAE buffer (total 6ul) and | ||
+ | load onto first well <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l18 level1 lfo6'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Mix 5ul of PCR | ||
+ | products with 1ul of loading dye and load onto wells.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l18 level1 lfo6'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Run gel at 90V | ||
+ | for 45minutes approximately<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l18 level1 lfo6'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Photograph gels | ||
+ | under UV light<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:14.0pt;line-height:160%'>Composite part ligation<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:14.0pt;line-height:160%'>The assembly of composite parts from | ||
+ | two existing <span class=SpellE>BioBricks</span> i.e. <span class=SpellE>BioBrick</span> | ||
+ | plasmid A (parent vector) and <span class=SpellE>BioBrick</span> plasmid B | ||
+ | (gene to be inserted) was performed through a restriction digest/ligation | ||
+ | protocol. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l3 level1 lfo7'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>200ng of each <span | ||
+ | class=SpellE>BioBrick</span> plasmid was digested as follows: Plasmid <span | ||
+ | class=SpellE>BioBrick</span> A with NEB restriction enzymes <span class=SpellE>SpeI</span> | ||
+ | and <span class=SpellE>PstI</span><span class=GramE>;</span> plasmid <span | ||
+ | class=SpellE>BioBrick</span> B with <span class=SpellE>XbaI</span> and <span | ||
+ | class=SpellE>PstI</span>, according to supplier protocol. (1hr @37<sup>o</sup>C, | ||
+ | then 20mins @80<sup>o</sup>C).<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l3 level1 lfo7'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>1µL of <span | ||
+ | class=GramE>Fast</span> alkaline <span class=SpellE>phosphatase</span> (Thermo | ||
+ | Scientific) was added to reaction tube of plasmid <span class=SpellE>BioBrick</span> | ||
+ | A to <span class=SpellE>dephosphorylate</span> the gene fragments and prevent | ||
+ | re-ligation of the parent vector. Reaction tubes were incubated at 37<sup>o</sup>C | ||
+ | for 60 mins, followed by a deactivation step at 80<sup>o</sup>C for 20 mins.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l3 level1 lfo7'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Ligation was then | ||
+ | performed with an insert to vector molar ratio of 3:1. 1µL of T4 ligase (NEB) | ||
+ | was added to the mix for ligation, according to supplier protocol. Ligation | ||
+ | reactions were performed at 37<sup>o</sup>C for 60 mins and kept on ice for | ||
+ | transformation into chemical competent cells.<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.j91d528avia></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>MANUAL TRYPSIN IN-GEL DIGESTION PROTOCOL FOR COOMASSIE STAINED GELS</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | class=SpellE><span style='font-size:14.0pt'>Coommassie</span></span><span | ||
+ | style='font-size:14.0pt'> de-stain gels: by washing briefly with 200µL NH4HCO3 | ||
+ | (Solution A) to make sure gel pieces are at the correct pH. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Add 200µL 50% <span | ||
+ | class=SpellE>Acetonitrile</span> / 50% 100mM NH4HCO3 (Solution B) into each | ||
+ | well. Vortex to mix and incubate for 10 minutes. Remove liquid.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Repeat step 2. | ||
+ | Gel pieces should be clear at this stage. If they are still blue, repeat as | ||
+ | necessary until <span class=SpellE>color</span> is gone.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Wash for 5 min | ||
+ | with 50µL of 100% <span class=SpellE>Acetonitrile</span> (Solution C) to | ||
+ | dehydrate gel pieces. Vortex during incubation.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Remove <span | ||
+ | class=SpellE>Acetonitrile</span>, <span class=GramE>then</span> let air-dry for | ||
+ | 10 min. The gel pieces should be noticeably shrunken and probably white.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Reduction and | ||
+ | Alkylation: Cover gel pieces with 50µL 10mM DTT in 50mM NH4HCO3 (Solution D). | ||
+ | Let proteins reduced for 45-60 min in 37<sup>o</sup>C incubator.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>7.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Remove DTT solution | ||
+ | and add 50µL of 55mM <span class=SpellE>iodoacetamide</span> in 50mM NH4HCO3 | ||
+ | (Solution E). Incubate for 45 min in dark place at room temperature.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>8.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span class=GramE><span style='font-size:14.0pt'>Remove | ||
+ | <span class=SpellE>iodoacetamide</span></span></span><span style='font-size: | ||
+ | 14.0pt'>, <span class=GramE>discard</span>.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>9.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Wash gel pieces | ||
+ | with 200ul of NH4HCO3 (Solution A) for 5 min with <span class=SpellE>vortexing</span>. | ||
+ | <span class=GramE>before</span> adding 100ul of 100% <span class=SpellE>Acetonitrile</span> | ||
+ | (Solution C).<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>10.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span class=GramE><span style='font-size:14.0pt'>Remove | ||
+ | liquid after 5 min</span></span><span style='font-size:14.0pt'>, <span | ||
+ | class=GramE>discard</span>.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>11.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Wash gel pieces | ||
+ | with 50ul 100mM NH4HCO3 (Solution A) for 10 min, then twice with 200ul 50% <span | ||
+ | class=SpellE>Acetonitrile</span> / 50% 100mM NH4HCO3 (Solution B) for 10min<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>12.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Dehydrate with | ||
+ | 100 <span class=SpellE>ul</span> 100% <span class=SpellE>Acetonitrile</span> | ||
+ | (Solution C) for 5 min as above.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>13.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Remove remaining | ||
+ | liquid and let the gel dried.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>14.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span class=SpellE><span style='font-size:14.0pt'>Trypsin</span></span><span | ||
+ | style='font-size:14.0pt'> Digestion: Prepare <span class=SpellE>trypsin</span> | ||
+ | mix to final concentration of 13ng/µL in 50mM Ammonium bicarbonate.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>15.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Add 30µL (or more | ||
+ | if required) of mixed <span class=SpellE>trypsin</span> solution to cover the | ||
+ | gel pieces. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>16.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Allow 30 min for | ||
+ | gel rehydration at 4<sup>o</sup>C (on ice). <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>17.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Digest overnight | ||
+ | at 37<sup>o</sup>C.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>18.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Peptide | ||
+ | Extraction: Transfer the digest solution supernatant (if any) into clean 0.65ml | ||
+ | <span class=SpellE>Eppendorf</span> tubes.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>19.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>To the gel | ||
+ | pieces, add 50 µL of 50% <span class=SpellE>acetonitrile</span> / 2% formic acid, | ||
+ | incubate 30 min. Spin, remove supernatant and combine with initial digest | ||
+ | solution supernatant. Please note that total volume may vary depending on the | ||
+ | gel sizes.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>20.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Vortex the | ||
+ | extracted digests, speed vac to reduce volume to 10 µL. If the remaining volume | ||
+ | is less than 10ul, use 2% formic acid to bring the volume up to 10 µL.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>21.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Spin at 14,000 | ||
+ | rpm for 30 min to remove any micro particulates. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>22.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Transfer the | ||
+ | supernatant to a fresh 0.65ml <span class=SpellE>eppendorf</span> tube for | ||
+ | storage at 4<sup>o</sup>C fridge OR directly into PCR plate for Mass spec for | ||
+ | analysis.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><o:p> </o:p></p> | ||
+ | |||
+ | <h4 style='text-align:justify;line-height:160%;mso-pagination:widow-orphan; | ||
+ | page-break-after:auto'><a name=h.tjzgdytzmhl9></a><b style='mso-bidi-font-weight: | ||
+ | normal'><span style='font-size:15.0pt;line-height:160%;font-family:Arial; | ||
+ | mso-fareast-font-family:Arial;mso-bidi-font-family:Arial;color:#333333; | ||
+ | text-decoration:none;text-underline:none'>Reagents </span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l12 level1 lfo9'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Buffer 100mM <b style='mso-bidi-font-weight:normal'>Ammonium | ||
+ | Bicarbonate </b><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:118.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l12 level2 lfo9'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>○<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>ADD 0.78 g ammonium bicarbonate to 100ml ddH20. Fill | ||
+ | first medium size reservoir.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l30 level1 lfo10'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Reduction: -<b style='mso-bidi-font-weight:normal'> <span | ||
+ | class=SpellE>Dithiothreitol</span> (DTT) </b><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l30 level2 lfo10'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>○<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Add 15 <span class=SpellE>mL</span> of 100 <span | ||
+ | class=SpellE>mM</span> ammonium bicarbonate to 23.1 mg of DTT to give a solution | ||
+ | of 10 <span class=SpellE>mM</span> DTT.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l14 level1 lfo11'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Alkylation <span class=GramE>-<span | ||
+ | style="mso-spacerun: yes"> </span><span class=SpellE><b style='mso-bidi-font-weight: | ||
+ | normal'>Iodoacetamide</b></span></span><b style='mso-bidi-font-weight:normal'>(DTT) | ||
+ | </b><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l14 level2 lfo11'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>○<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Add 15 <span class=SpellE>mL</span> of 100 <span | ||
+ | class=SpellE>mM</span> ammonium bicarbonate to 153 mg of <span class=SpellE>iodoacetamide</span> | ||
+ | to give a solution of 55 <span class=SpellE>mM</span> <span class=SpellE>iodoacetamide</span> | ||
+ | - 15 <span class=SpellE>mL</span> of each is enough for up to 96 samples.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l5 level1 lfo12'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Peptide Extraction Solution -<b style='mso-bidi-font-weight: | ||
+ | normal'> 2% Formic acid/ 50% <span class=SpellE>Acetonitrile</span> solution </b><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l5 level2 lfo12'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>○<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Add 2000µL of formic acid and 50 <span class=SpellE>mL</span> | ||
+ | of <span class=SpellE>acetonitrile</span> to 75 <span class=SpellE>mL</span> of | ||
+ | ddH2O. 15 <span class=SpellE>mL</span> is enough for up to 96 samples.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l24 level1 lfo13'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Dehydration -<b style='mso-bidi-font-weight:normal'> <span | ||
+ | class=SpellE>Acetonitrile</span> </b><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l24 level2 lfo13'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>○<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>A full square reservoir is plenty for up to 96 samples | ||
+ | with dehydration and/or <span class=SpellE>Coomassie</span> <span class=SpellE>destaining</span>.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l27 level1 lfo14'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | class=SpellE><b style='mso-bidi-font-weight:normal'><span style='font-size: | ||
+ | 14.0pt'>Trypsin</span></b></span><span style='font-size:14.0pt'><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l27 level2 lfo14'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>○<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | class=SpellE><span style='font-size:14.0pt'>Trypsin</span></span><span | ||
+ | style='font-size:14.0pt'> is prepared by adding 200µL of <span class=SpellE>Resuspension</span> | ||
+ | Buffer into one vial containing 20ug of Proteomics grade <span class=SpellE>trypsin</span>. | ||
+ | <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l27 level2 lfo14'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>○<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Add 1.2ml of 50mM NH4HCO3 into each <span | ||
+ | class=SpellE>trypsin</span> vial (final concentration of <span class=SpellE>Trypsin</span> | ||
+ | is 16ng/µL). Mix thoroughly. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:118.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l27 level2 lfo14'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>○<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>This will provide enough <span class=SpellE>Trypsin</span> | ||
+ | for 46 digestions.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><o:p> </o:p></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><o:p> </o:p></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt'>Mass Spec<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Samples need to | ||
+ | be spotted on the 384 spot MALDI plate: Odd rows are used for samples, and even | ||
+ | rows are for calibration standards. It is important that one sample spot in the | ||
+ | odd row is coupled by a calibration spot in the even row.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Samples are | ||
+ | cleaned up with C18 Zip tips before spotting onto the plate. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Start with a | ||
+ | fresh <span class=SpellE>Ziptip</span>, using a 10 µL pipette, and slowly | ||
+ | withdraw and dispense to waste 10 µL 90% (v/v) <span class=SpellE>acetonitrile</span> | ||
+ | / 0.1 % TFA three times. Do not throw away the tip!<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Slowly withdraw | ||
+ | and dispense to waste 10 µL 0.1 % TFA five times to equilibrate the C18 | ||
+ | material in aqueous buffer.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Slowly withdraw | ||
+ | and dispense your first sample (10 µL) ten times. After this step the peptides | ||
+ | from your digest are bound onto the C18 resin in the tip.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Withdraw and | ||
+ | discard to waste 10 µL of 0.1 % TFA five times. This washes away unwanted salts | ||
+ | and contaminants. Make sure you expel all the liquid on the last wash.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: yes"> | ||
+ | </span>7. Withdraw 1 µL of matrix solution. This contains 70% <span | ||
+ | class=SpellE>acetonitrile</span> so it is<span style="mso-spacerun: | ||
+ | yes"> </span>used to elute peptides in the presence of | ||
+ | matrix. <span class=GramE>(4 mg / <span class=SpellE>mL</span> CHCA | ||
+ | [α-cyano-4-hydroxycynnamic acid], 70 % <span class=SpellE>MeCN</span>, 0.1 | ||
+ | % TFA).</span> <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: | ||
+ | yes"> </span>8. Spot the eluted peptide sample in | ||
+ | matrix solution directly onto the MALDI target plate in an odd row, trying not | ||
+ | to touch the surface of the plate with the tip. Record the location of your | ||
+ | samples.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: | ||
+ | yes"> </span>9. New C18 tip is needed for | ||
+ | each sample. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: yes"> | ||
+ | </span>10. BSA digest at 1pmol/µL is used as control. This does not required <span | ||
+ | class=SpellE>ziptipping</span>.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: | ||
+ | yes"> </span>11. Next, prepare calibration | ||
+ | standards (<span class=SpellE>Pepmix</span> and Matrix solutions at 1:1 ratio), | ||
+ | and spot 1ul of this onto spots in even row next to your samples. These are | ||
+ | your near-point calibration standards. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: | ||
+ | yes"> </span>12.Allow the plate to | ||
+ | air-dry.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: | ||
+ | yes"> </span>13. Plates can be stored in 4<sup>o</sup>C | ||
+ | fridge and in the dark for up to a week, ready for analysis.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: | ||
+ | yes"> </span>14. Samples are <span | ||
+ | class=SpellE>analyzed</span> using the 4800 Plus MALDI <span class=SpellE>Tof-Tof</span> | ||
+ | <span class=SpellE>Analyzer</span>.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal><o:p> </o:p></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.eh11zdoajzzn></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>LB AGAR PLATE PREPARATION</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l28 level1 lfo16'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Add 15g <span class=SpellE>Bacto</span> agar to 1000mL | ||
+ | of LB media and autoclave (121<sup>o</sup>C for 15mins).<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l28 level1 lfo16'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Add 1000uL of <span | ||
+ | class=SpellE>Chloroamphenicol</span> (25mg/mL), Ampicillin (50mg/mL) or <span | ||
+ | class=SpellE>Kanamycin</span> (30mg/mL) and mix well before plating out and | ||
+ | setting the agar.<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.8d6a21bkpjv7></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>LB MEDIA</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l22 level1 lfo17'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span | ||
+ | style='font-size:14.0pt'> <span class=SpellE>Tryptone</span> 10g, Yeast extract | ||
+ | 5g, <span class=SpellE>NaCl</span> 10g, <span class=SpellE>Milli</span>-Q water | ||
+ | to 1000mL.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l22 level1 lfo17'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span | ||
+ | style='font-size:14.0pt'> Dissolved 10g <span class=SpellE>tryptone</span>, 5g | ||
+ | yeast extract and 10g <span class=SpellE>NaCl</span> in 800mL <span | ||
+ | class=SpellE>Milli</span>-Q water, making use of a magnetic stirrer. Once | ||
+ | dissolved, bring volume up to 1 L using <span class=SpellE>Milli</span>-Q | ||
+ | water. Autoclave 1L of the solution (121<sup>o</sup>C, 15 min, standard liquid | ||
+ | cycle). <o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.msyjoqpi6c3m></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>SOC MEDIA (FOR COMPETENT CELLS)</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l20 level1 lfo18'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span | ||
+ | style='font-size:14.0pt'> 10g <span class=SpellE>bacto-tryptone</span>, 2.5g <span | ||
+ | class=SpellE>bacto</span>-yeast, 1000µl 5M <span class=SpellE>NaCl</span>, | ||
+ | 417µL 3M <span class=SpellE>KCl</span>, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose | ||
+ | & 500mL <span class=SpellE>Milli</span>-Q water. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l20 level1 lfo18'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span | ||
+ | style='font-size:14.0pt'> The adjusted quantities were combined in a 1 L | ||
+ | measuring column with constant stirring and then placed in the autoclave for | ||
+ | sterilisation (121<sup>o</sup>C, 15 min, standard liquid cycle).<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.q141kogjduod></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>NANODROP for THERMO SCIENTIFIC <span class=SpellE>NanoDrop</span> 2000 | ||
+ | Spectrophotometer</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l9 level1 lfo19'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Pipette 1-2µl of blanking buffer onto the bottom | ||
+ | pedestal, lower the arm and click the BLANK button. The blank solution is the | ||
+ | same buffer that the DNA of interest is dissolved in.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddle style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l9 level1 lfo19'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Wipe away the | ||
+ | blank. Pipette 1-2µl of DNA sample and hit MEASURE<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l9 level1 lfo19'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Wipe away the | ||
+ | last sample to prepare for the next sample, and repeat.<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.df10up6a0vzh></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>PCR Mixture: General recipe for PCR is as follows:</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l11 level1 lfo20'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>4 µL of 5x <span class=SpellE>phusion</span> buffer.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l11 level1 lfo20'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>0.4 µL <span class=SpellE>dNTPs</span>.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l11 level1 lfo20'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>0.6 µL of DMSO.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l11 level1 lfo20'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:13.5pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>0.2 µL of polymerase</span><span style='font-size: | ||
+ | 13.5pt'>.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l11 level1 lfo20'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>11.8 µL of water.<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.6eeyqdi9mhqi></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>To this mixture, add:</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l8 level1 lfo21'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>1 µL of forward primer.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l8 level1 lfo21'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>1 µL of reverse primer.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l8 level1 lfo21'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>1 µL of template.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l8 level1 lfo21'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Total volume = 20 µL<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.ka6u2m955lhu></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>PCR settings: A general program for PCR is as follows:</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l4 level1 lfo22'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Initial <span class=SpellE>denaturation</span> at 98<sup>o</sup>C | ||
+ | for 30seconds.<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.xq8hqhbk6lk5></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>Followed by 30 repeats of:</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l32 level1 lfo23'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | class=SpellE><span style='font-size:14.0pt'>Denaturation</span></span><span | ||
+ | style='font-size:14.0pt'> – 98<sup>o</sup>C for 10seconds.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l32 level1 lfo23'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Annealing – 60<sup>o</sup>C for 10seconds.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l32 level1 lfo23'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Extension – 72<sup>o</sup>C for 2minutes.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l32 level1 lfo23'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Final extension – 72<sup>o</sup>C for 10minutes.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt'>Plasmid Prep<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge | ||
+ | @13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL <span | ||
+ | class=SpellE>Eppendorf’s</span><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Discard | ||
+ | supernatant and add 1.9 <span class=SpellE>mL</span> of culture and centrifuge | ||
+ | again @13,200 rpm for 10 min<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Re-suspend <span | ||
+ | class=SpellE>pelleted</span> cells in P1 Buffer (250µl) <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Add 250µL of P2 | ||
+ | buffer & invert 4-6 times (turns homogenous blue)<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Add 350µL of N3 | ||
+ | & mix by inverting (turns colourless)<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge @ | ||
+ | 10min 13,000 rpm to obtain a pellet<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>7.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Transfer | ||
+ | supernatant in QIA Prep Spin Column by <span class=SpellE>pipetting</span><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>8.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge 30-60 | ||
+ | sec - discard flow through<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>9.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Wash QIA Prep | ||
+ | Spin Column with 0.5mL of PB<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>10.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge for | ||
+ | 30-60 seconds - discard flow through<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>11.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Wash spin column | ||
+ | by adding 0.75 <span class=SpellE>mL</span> PE buffer<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>12.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge for | ||
+ | 30-60 seconds - discard flow through and centrifuge for a further 1min<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>13.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Place <span | ||
+ | class=SpellE>QIAPrep</span> Column in clean <span class=SpellE>Eppendorf</span><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>14.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Elute DNA add | ||
+ | 50µl of water, stand for 1min, centrifuge for 1min<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>15.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>QIA prep - Spin <span | ||
+ | class=SpellE>miniprep</span> buffer<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><o:p> </o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><o:p> </o:p></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt'>SDS-PAGE<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal><o:p> </o:p></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Re-suspend <span | ||
+ | class=SpellE>pelleted</span> bacterial cells in 200µL of <span class=SpellE>Milli</span>-Q-water<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Transfer 50 µL of | ||
+ | suspension into new <span class=SpellE>Eppendorf</span> tubes and combine with | ||
+ | 50ul of 2xTruSep sample buffer.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'><span | ||
+ | style="mso-spacerun: yes"> </span>Shear the cells using a Hamilton | ||
+ | syringe.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge the | ||
+ | preparation for 3minutes @ 13,000 rpm.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Load 20 µL of the | ||
+ | supernatant into gel.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Conduct | ||
+ | electrophoresis at a constant voltage (200V) for 1 hour.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>7.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span class=SpellE><span style='font-size:14.0pt'>Coommassie</span></span><span | ||
+ | style='font-size:14.0pt'> Stain for ~30 minutes.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><o:p> </o:p></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><o:p> </o:p></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt'>Heat Shock<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Obtain competent | ||
+ | cells from -80<sup>o</sup>C.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Defrost gently on | ||
+ | ice. 100µl is sufficient for 2 transformations.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Add 1-10µl of | ||
+ | plasmid DNA/ ligation mix to each tube. Incubate on ice for 5 min.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Put the tubes in | ||
+ | the 42<sup>o</sup>C water bath for 30 seconds, <span class=GramE>then</span> | ||
+ | back on ice for 2 min.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Add 200µl of SOC | ||
+ | media to each tube, and incubate in the 37<sup>o</sup>C shaker for 10 min or up | ||
+ | to 1h (10min for purified plasmid or 1h for ligation mix).<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>For each tube of | ||
+ | cells, spread 50µl onto one LB plate with appropriate antibiotic, and 500µl | ||
+ | onto a second plate, using aseptic technique. Place your plate upside-down in | ||
+ | the 37<sup>o</sup>C incubator.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><o:p> </o:p></span></p> | ||
+ | |||
+ | <h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a | ||
+ | name=h.uwg4fet686y3></a><b style='mso-bidi-font-weight:normal'><span | ||
+ | style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline: | ||
+ | none'>Induction of <span class=SpellE>Operons</span></span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align: | ||
+ | justify;text-indent:-18.0pt;line-height:normal;mso-list:l13 level1 lfo27'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'>Re-transform restriction-digest screened plasmids | ||
+ | according to the heat shock transformation protocol.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l13 level1 lfo27'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Screen plates for | ||
+ | transformant colonies and place in 5mL of ZYM-5052 broth with appropriate | ||
+ | antibiotic concentration at 37<sup>o</sup>C with shaking until OD600¬ is 2.0 | ||
+ | – 3.0.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;mso-list:l13 level1 lfo27'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Take whole volume | ||
+ | and place in 45mL of ZYM-5052 broth in a sterile conical flask containing | ||
+ | appropriate antibiotic concentration. Incubate at 37<sup>o</sup>C with shaking.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l13 level1 lfo27'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Cell pellets were | ||
+ | collected through centrifugation at 12000 rpm for 5 mins. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l13 level1 lfo27'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family: | ||
+ | Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Cell pellets were | ||
+ | resuspended in re-suspension buffer (Containing: 10% glycerol w/v, 50mM <span | ||
+ | class=SpellE>Tricene</span> <span class=SpellE>NaOH</span> pH 8.0, 2mM MgCl<sub>2</sub>, | ||
+ | 1mM DTT) and whole lysate was collected through a French Press for functional | ||
+ | assays of <span class=SpellE>operon</span> protein products in lysate.<o:p></o:p></span></p> | ||
+ | |||
+ | <h4 style='text-align:justify;line-height:160%;mso-pagination:widow-orphan; | ||
+ | page-break-after:auto'><a name=h.s0f83usywiy1></a><span class=SpellE><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt;line-height: | ||
+ | 160%;font-family:Arial;mso-fareast-font-family:Arial;mso-bidi-font-family:Arial; | ||
+ | color:#333333;text-decoration:none;text-underline:none'>MgPE</span></b></span><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt;line-height: | ||
+ | 160%;font-family:Arial;mso-fareast-font-family:Arial;mso-bidi-font-family:Arial; | ||
+ | color:#333333;text-decoration:none;text-underline:none'> enzymatic functional | ||
+ | assay</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-indent: | ||
+ | -18.0pt;mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size: | ||
+ | 14.0pt;line-height:115%'><span style='mso-list:Ignore'>1.<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:115%'>Collect whole lysate, from induced <span | ||
+ | class=SpellE>operon</span> 3 containing <span class=SpellE>ChlM</span> | ||
+ | expressed proteins, using <span class=SpellE>ChlM</span> <span class=SpellE>pET</span> | ||
+ | (L. <span class=SpellE>Meinecke</span> et <span class=GramE>al.2010)as</span> a | ||
+ | negative control.<span style="mso-spacerun: yes"> </span><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-top:.1pt;margin-right:0cm; | ||
+ | margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt; | ||
+ | mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt; | ||
+ | mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span | ||
+ | style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Prepare a master | ||
+ | mix of recombinant <span class=SpellE>ChlM</span>, 25µl of S-<span | ||
+ | class=SpellE>Adenosyl</span>-L- <span class=SpellE>methionine</span> and 65µl | ||
+ | of 2µM of Mg-<span class=SpellE>protoporphyrin</span> IX.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-top:.1pt;margin-right:0cm; | ||
+ | margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt; | ||
+ | mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt; | ||
+ | mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span | ||
+ | style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Incubate at 30</span><span | ||
+ | style='font-size:14.0pt;font-family:"Noteworthy Light";mso-bidi-font-family: | ||
+ | "Noteworthy Light"'>℃</span><span style='font-size:14.0pt'> with gentle | ||
+ | agitation for 8-10min<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-top:.1pt;margin-right:0cm; | ||
+ | margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt; | ||
+ | mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt; | ||
+ | mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span | ||
+ | style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>100µL of acetone | ||
+ | was added to precipitate protein. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-top:.1pt;margin-right:0cm; | ||
+ | margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt; | ||
+ | mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt; | ||
+ | mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span | ||
+ | style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>The sample was | ||
+ | spun down, to separate the protein and supernatant.<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddleCxSpLast style='margin-top:.1pt;margin-right:0cm; | ||
+ | margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt; | ||
+ | mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt; | ||
+ | mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span | ||
+ | style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'> | ||
+ | </span></span></span><![endif]><span style='font-size:14.0pt'>Supernatant was | ||
+ | then subjected to UHPLC to and observed peaks. <o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal><span style='font-size:14.0pt;line-height:115%'><o:p> </o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal><span style='font-size:14.0pt;line-height:115%'><span | ||
+ | style="mso-spacerun: yes"> </span></span></p> | ||
+ | |||
+ | <h4 style='text-align:justify;line-height:160%;mso-pagination:widow-orphan; | ||
+ | page-break-after:auto'><a name=h.ynrtu1oxkw8d></a><b style='mso-bidi-font-weight: | ||
+ | normal'><span style='font-size:15.0pt;line-height:160%;font-family:Arial; | ||
+ | mso-fareast-font-family:Arial;mso-bidi-font-family:Arial;color:#333333; | ||
+ | text-decoration:none;text-underline:none'>UHPLC protocol</span></b></h4> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:14.0pt;line-height:160%'>The UHPLC protocol was performed | ||
+ | according to <span class=SpellE>Sawicki</span> and Willows (2007). Briefly, a | ||
+ | Shimadzu HPLC system was used at 2ml/min with an <span class=SpellE>Alltech</span> | ||
+ | C8 column. A 5 min linear gradient was used for separation of Mg-<span | ||
+ | class=SpellE>protoporphyrin</span> IX and Magnesium-<span class=SpellE>protoporphyrin</span> | ||
+ | IX mono-methyl ester. Compounds were detected at an excitation wavelength of | ||
+ | excitation of 416nm and emission of 595nm.<span style="mso-spacerun: | ||
+ | yes"> </span><o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt;line-height: | ||
+ | 160%'><o:p> </o:p></span></b></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><b | ||
+ | style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt;line-height: | ||
+ | 160%'>Auto induction recipe <o:p></o:p></span></b></p> | ||
+ | |||
+ | <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 | ||
+ | style='border-collapse:collapse;border:none;mso-border-alt:solid black; | ||
+ | mso-border-themecolor:text1;mso-border-alt:.5pt;mso-yfti-tbllook:191; | ||
+ | mso-padding-alt:0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid black; | ||
+ | mso-border-insideh-themecolor:text1;mso-border-insidev:.5pt solid black; | ||
+ | mso-border-insidev-themecolor:text1'> | ||
+ | <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'> | ||
+ | <td width=239 valign=top style='width:239.4pt;border:solid white;mso-border-themecolor: | ||
+ | background1;border:1.0pt;border-bottom:none;mso-border-top-alt:solid white; | ||
+ | mso-border-top-themecolor:background1;mso-border-top-alt:.5pt;mso-border-left-alt: | ||
+ | solid white;mso-border-left-themecolor:background1;mso-border-left-alt:.5pt; | ||
+ | mso-border-right-alt:solid white;mso-border-right-themecolor:background1; | ||
+ | mso-border-right-alt:.5pt;background:#FEFEFE;mso-shading:windowtext; | ||
+ | mso-pattern:solid white;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:14.0pt;line-height:160%'>1.ZYM-5052 Medium <o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l1 level1 lfo29'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>ZY media 9.57ml <o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l1 level1 lfo29'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>1m MgSO4 20ul<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l1 level1 lfo29'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>50x5052 200ul<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l1 level1 lfo29'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>50 x M 200ul<o:p></o:p></span></p> | ||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:15.0pt;line-height:160%'><o:p> </o:p></span></p> | ||
+ | </td> | ||
+ | <td width=239 valign=top style='width:239.4pt;border:solid white;mso-border-themecolor: | ||
+ | background1;border:1.0pt;border-left:none;mso-border-left-alt:solid white; | ||
+ | mso-border-left-themecolor:background1;mso-border-left-alt:.5pt;mso-border-alt: | ||
+ | solid white;mso-border-themecolor:background1;mso-border-alt:.5pt;padding: | ||
+ | 0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:14.0pt;line-height:160%'>2. 50 x M<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>DI water 80ml<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>Na2HPO4 17.75g<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>KH2PO4 17g<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>NH4Cl 13.4<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>Na2SO4 3.55g<o:p></o:p></span></p> | ||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:15.0pt;line-height:160%'><o:p> </o:p></span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr style='mso-yfti-irow:1;mso-yfti-lastrow:yes'> | ||
+ | <td width=239 valign=top style='width:239.4pt;border:solid white;mso-border-themecolor: | ||
+ | background1;border:1.0pt;border-top:none;mso-border-left-alt:solid white; | ||
+ | mso-border-left-themecolor:background1;mso-border-left-alt:.5pt;mso-border-bottom-alt: | ||
+ | solid white;mso-border-bottom-themecolor:background1;mso-border-bottom-alt: | ||
+ | .5pt;mso-border-right-alt:solid white;mso-border-right-themecolor:background1; | ||
+ | mso-border-right-alt:.5pt;padding:0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:14.0pt;line-height:160%'>3. 50 x 5052<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l6 level1 lfo31'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>Glycerol 25g (weigh in Beaker)<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l6 level1 lfo31'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>DI water 73ml<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l6 level1 lfo31'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>Glucose 2.5g<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l6 level1 lfo31'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | class=GramE><span style='font-size:14.0pt;line-height:160%'>alpha</span></span><span | ||
+ | style='font-size:14.0pt;line-height:160%'> lactose 10g<o:p></o:p></span></p> | ||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:15.0pt;line-height:160%'><o:p> </o:p></span></p> | ||
+ | </td> | ||
+ | <td width=239 valign=top style='width:239.4pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid white;mso-border-bottom-themecolor:background1; | ||
+ | border-bottom:1.0pt;border-right:solid white;mso-border-right-themecolor: | ||
+ | background1;border-right:1.0pt;mso-border-top-alt:solid white;mso-border-top-themecolor: | ||
+ | background1;mso-border-top-alt:.5pt;mso-border-left-alt:solid white; | ||
+ | mso-border-left-themecolor:background1;mso-border-left-alt:.5pt;mso-border-alt: | ||
+ | solid white;mso-border-themecolor:background1;mso-border-alt:.5pt;padding: | ||
+ | 0cm 5.4pt 0cm 5.4pt'> | ||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:14.0pt;line-height:160%'>4. 1M MgSO4<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l26 level1 lfo32'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>MgSO4.7H2O 24.65g<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l26 level1 lfo32'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>DI water 87ml <o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l26 level1 lfo32'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>5. ZY Media (400mL)<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l19 level1 lfo33'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | class=SpellE><span style='font-size:14.0pt;line-height:160%'>Trypton</span></span><span | ||
+ | style='font-size:14.0pt;line-height:160%'> 4g<o:p></o:p></span></p> | ||
+ | <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space: | ||
+ | auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l19 level1 lfo33'><![if !supportLists]><span | ||
+ | style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times; | ||
+ | mso-bidi-font-family:Times'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt;line-height:160%'>Yeast extract 2g <o:p></o:p></span></p> | ||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:15.0pt;line-height:160%'><o:p> </o:p></span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:160%'><span | ||
+ | style='font-size:15.0pt;line-height:160%'><o:p> </o:p></span></p> | ||
+ | |||
+ | <h3 style='text-align:justify;line-height:160%;mso-pagination:widow-orphan; | ||
+ | page-break-after:auto'><a name=h.2rdkrjouyz6k></a><span style='font-size:14.0pt; | ||
+ | line-height:160%;font-family:Arial;mso-fareast-font-family:Arial;mso-bidi-font-family: | ||
+ | Arial;color:black;font-weight:normal'><o:p> </o:p></span></h3> | ||
+ | |||
+ | <h3 style='text-align:justify;line-height:160%;mso-pagination:widow-orphan; | ||
+ | page-break-after:auto'><span style='font-size:19.5pt;line-height:160%; | ||
+ | font-family:Arial;mso-fareast-font-family:Arial;mso-bidi-font-family:Arial; | ||
+ | color:windowtext'>References</span></h3> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><o:p> </o:p></p> | ||
+ | |||
+ | <p class=normalcxspmiddle style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l15 level1 lfo34'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | class=SpellE><span class=GramE><span style='font-size:14.0pt'>sawicki,A</span></span><span | ||
+ | style='font-size:14.0pt'>.,Willows,R.D</span></span><span style='font-size: | ||
+ | 14.0pt'>.(2007).S-<span class=SpellE>Adenosyl-L-methionine:magnesium-protoporphyrin</span> | ||
+ | IX O-<span class=SpellE>methyltransferase</span> from <span class=SpellE>Rhodebacter</span> | ||
+ | <span class=SpellE>capsulatus</span>: mechanistic insights and stimulation with | ||
+ | phospholipids. Journal of Biochemistry, 406, 469-478. <span class=SpellE><span | ||
+ | class=GramE>doi</span></span>: 10.1042/BJ20070284<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><o:p> </o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddle style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l15 level1 lfo34'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: yes"> </span><span | ||
+ | class=SpellE>William<span class=GramE>,F</span></span>.,<span | ||
+ | style="mso-spacerun: yes"> </span>Studier. (2014). Stable Expression | ||
+ | Clones and Auto-Induction for Protein Production in <i style='mso-bidi-font-style: | ||
+ | normal'>E.coli. </i>Structural Genomics: General Applications, Methods in | ||
+ | Molecular Biology.1091, 17-32 DOI 10.1007/978-1-62703-691-7_2<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><span | ||
+ | style='font-size:14.0pt'><o:p> </o:p></span></p> | ||
+ | |||
+ | <p class=normalcxspmiddle style='margin-left:59.0pt;mso-add-space:auto; | ||
+ | text-align:justify;text-indent:-18.0pt;mso-list:l15 level1 lfo34'><![if !supportLists]><span | ||
+ | style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family: | ||
+ | Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>●<span | ||
+ | style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span | ||
+ | class=SpellE><span style='font-size:14.0pt'>Meinecke</span></span><span | ||
+ | style='font-size:14.0pt'>, L., <span class=SpellE>Alawady</span>, A., <span | ||
+ | class=SpellE>Schroda</span>, M., Willows, R., Kobayashi, M.C<span class=GramE>,.</span> | ||
+ | <span class=SpellE>Niyogi</span>, K.K<span class=GramE>,.</span> Grimm, B., | ||
+ | Beck, C.F. (2010). Chlorophyll-deficient mutants of <span class=SpellE>Chlamydomonas</span> | ||
+ | <span class=SpellE>reinhardtii</span> that accumulate magnesium <span | ||
+ | class=SpellE>protoporphyrin</span> IX. <span class=SpellE><span class=GramE>doi</span></span>: | ||
+ | 10.1007/s11103-010-9604-9<o:p></o:p></span></p> | ||
+ | |||
+ | <p class=MsoNormal style='text-align:justify;line-height:normal'><u><span | ||
+ | style='font-size:14.0pt'><span style="mso-spacerun: yes"> </span></span></u><span | ||
+ | style='font-size:14.0pt'><o:p></o:p></span></p> | ||
+ | |||
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Revision as of 05:54, 18 September 2015
Experiments & Protocols
![](https://static.igem.org/mediawiki/2015/a/ab/MQAust_OverView_FlowChart.png)
This page is for descriptions of the experiments, research and protocols we used in our iGEM project.
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of our project
Inspiration
Protocols
Making competent cells
1.
Using a sterile
plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate.
Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22oC,
200-250rpm.
2.
A600 should be
0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~
0.5.
3. Remove the flask from the incubator and place on ice
for 10 minutes.
4. Transfer the culture to a 15mL centrifuge tube and
spin at 2500 x g for 10 min at 4oC
5. Pour off and discard the supernatant, and immediately
place the tube on ice.
6. Resuspend your cells in 1mL of ice-cold TB buffer,
make sure there are no clumps of cells left, but also treat your cells gently
and keep them cold.
7. Add ice-cold TB buffer to bring volume up to 1/5th of
the original culture volume (~30mL in this case). Mix the tube by gently
inverting 3 times.
8. Incubate the tube on ice for 10 minutes.
9. Centrifuge at 2,500 x g for 7
minutes at 4oC, discard the supernatant.
10.
Gently resuspend
the cells in ~1/20th of the original culture volume of ice-cold TB buffer.
NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. e.g. if the culture OD600 was 0.1 then resuspend in 1/100th
of original volume.
11.
Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension,
keeping the remainder on ice in the 15mL tube.
12.
Add 70µl of DMSO
to the 930µl of cell suspension. Mix gently by swirling, and place on ice.
13.
Aliquot 100µl of
the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the
tubes with: Date – Strain. Snap freeze with
liquid nitrogen.
14.
Repeat step 11-13
for the rest of your cell suspension in step 10. Store cells at -80oC
TB BUFFER
● Ingredients: 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl.
● Methods: All components (except for MnCl2-4H2O) were mixed and
dissolved in 500 mL of water and pH adjusted to 6.7
with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL
of water, mixed and solution adjusted to 1 L. Sterilisation via filtration
followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.
EDTA BUFFER
● Ingredients: 37.22g EDTA solid, 180 mL
of water and pH adjusted to 8.0 using 10M NaOH.
● Methods: Components were combined then pH adjusted.
TAE BUFFER
● Ingredients: 121.2g Tris base (dissolved
in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0).
● Methods: A total volume of 500 mL
was made up as a 50x stock solution using all components
Making agarose gel
Preparing the Gel
1. Mix 1g of agarose powder with 100ml of 1x TAE buffer
and heat for 1minute or until all agarose is dissolved.
2.
Wait until it has
cooled (not set), and add 1ul of GelRed into the
mixture.
3.
Pour the solution
into a cast with an appropriate comb.
4.
Leave to set.
Running the Gel
1. Mix 1ul of 1kbp DNA ladder with 6ul of loading dye (bromophenol blue) and 4ul of 1x TAE buffer (total 6ul) and
load onto first well
2.
Mix 5ul of PCR
products with 1ul of loading dye and load onto wells.
3.
Run gel at 90V
for 45minutes approximately
4.
Photograph gels
under UV light
Composite part ligation
The assembly of composite parts from
two existing BioBricks i.e. BioBrick
plasmid A (parent vector) and BioBrick plasmid B
(gene to be inserted) was performed through a restriction digest/ligation
protocol.
1.
200ng of each BioBrick plasmid was digested as follows: Plasmid BioBrick A with NEB restriction enzymes SpeI
and PstI; plasmid BioBrick B with XbaI and PstI, according to supplier protocol. (1hr @37oC,
then 20mins @80oC).
2.
1µL of Fast alkaline phosphatase (Thermo
Scientific) was added to reaction tube of plasmid BioBrick
A to dephosphorylate the gene fragments and prevent
re-ligation of the parent vector. Reaction tubes were incubated at 37oC
for 60 mins, followed by a deactivation step at 80oC for 20 mins.
3.
Ligation was then
performed with an insert to vector molar ratio of 3:1. 1µL of T4 ligase (NEB)
was added to the mix for ligation, according to supplier protocol. Ligation
reactions were performed at 37oC for 60 mins and kept on ice for
transformation into chemical competent cells.
MANUAL TRYPSIN IN-GEL DIGESTION PROTOCOL FOR COOMASSIE STAINED GELS
1. Coommassie de-stain gels: by washing briefly with 200µL NH4HCO3
(Solution A) to make sure gel pieces are at the correct pH.
2.
Add 200µL 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) into each
well. Vortex to mix and incubate for 10 minutes. Remove liquid.
3.
Repeat step 2.
Gel pieces should be clear at this stage. If they are still blue, repeat as
necessary until color is gone.
4.
Wash for 5 min
with 50µL of 100% Acetonitrile (Solution C) to
dehydrate gel pieces. Vortex during incubation.
5.
Remove Acetonitrile, then let air-dry for
10 min. The gel pieces should be noticeably shrunken and probably white.
6.
Reduction and
Alkylation: Cover gel pieces with 50µL 10mM DTT in 50mM NH4HCO3 (Solution D).
Let proteins reduced for 45-60 min in 37oC incubator.
7.
Remove DTT solution
and add 50µL of 55mM iodoacetamide in 50mM NH4HCO3
(Solution E). Incubate for 45 min in dark place at room temperature.
8.
Remove
iodoacetamide, discard.
9.
Wash gel pieces
with 200ul of NH4HCO3 (Solution A) for 5 min with vortexing.
before adding 100ul of 100% Acetonitrile
(Solution C).
10.
Remove
liquid after 5 min, discard.
11.
Wash gel pieces
with 50ul 100mM NH4HCO3 (Solution A) for 10 min, then twice with 200ul 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) for 10min
12.
Dehydrate with
100 ul 100% Acetonitrile
(Solution C) for 5 min as above.
13.
Remove remaining
liquid and let the gel dried.
14.
Trypsin Digestion: Prepare trypsin
mix to final concentration of 13ng/µL in 50mM Ammonium bicarbonate.
15.
Add 30µL (or more
if required) of mixed trypsin solution to cover the
gel pieces.
16.
Allow 30 min for
gel rehydration at 4oC (on ice).
17.
Digest overnight
at 37oC.
18.
Peptide
Extraction: Transfer the digest solution supernatant (if any) into clean 0.65ml
Eppendorf tubes.
19.
To the gel
pieces, add 50 µL of 50% acetonitrile / 2% formic acid,
incubate 30 min. Spin, remove supernatant and combine with initial digest
solution supernatant. Please note that total volume may vary depending on the
gel sizes.
20.
Vortex the
extracted digests, speed vac to reduce volume to 10 µL. If the remaining volume
is less than 10ul, use 2% formic acid to bring the volume up to 10 µL.
21.
Spin at 14,000
rpm for 30 min to remove any micro particulates.
22.
Transfer the
supernatant to a fresh 0.65ml eppendorf tube for
storage at 4oC fridge OR directly into PCR plate for Mass spec for
analysis.
Reagents
● Buffer 100mM Ammonium
Bicarbonate
○ ADD 0.78 g ammonium bicarbonate to 100ml ddH20. Fill
first medium size reservoir.
● Reduction: - Dithiothreitol (DTT)
○ Add 15 mL of 100 mM ammonium bicarbonate to 23.1 mg of DTT to give a solution
of 10 mM DTT.
● Alkylation - Iodoacetamide(DTT)
○ Add 15 mL of 100 mM ammonium bicarbonate to 153 mg of iodoacetamide
to give a solution of 55 mM iodoacetamide
- 15 mL of each is enough for up to 96 samples.
● Peptide Extraction Solution - 2% Formic acid/ 50% Acetonitrile solution
○ Add 2000µL of formic acid and 50 mL
of acetonitrile to 75 mL of
ddH2O. 15 mL is enough for up to 96 samples.
● Dehydration - Acetonitrile
○ A full square reservoir is plenty for up to 96 samples
with dehydration and/or Coomassie destaining.
● Trypsin
○ Trypsin is prepared by adding 200µL of Resuspension
Buffer into one vial containing 20ug of Proteomics grade trypsin.
○ Add 1.2ml of 50mM NH4HCO3 into each trypsin vial (final concentration of Trypsin
is 16ng/µL). Mix thoroughly.
○ This will provide enough Trypsin
for 46 digestions.
Mass Spec
1.
Samples need to
be spotted on the 384 spot MALDI plate: Odd rows are used for samples, and even
rows are for calibration standards. It is important that one sample spot in the
odd row is coupled by a calibration spot in the even row.
2.
Samples are
cleaned up with C18 Zip tips before spotting onto the plate.
3.
Start with a
fresh Ziptip, using a 10 µL pipette, and slowly
withdraw and dispense to waste 10 µL 90% (v/v) acetonitrile
/ 0.1 % TFA three times. Do not throw away the tip!
4.
Slowly withdraw
and dispense to waste 10 µL 0.1 % TFA five times to equilibrate the C18
material in aqueous buffer.
5.
Slowly withdraw
and dispense your first sample (10 µL) ten times. After this step the peptides
from your digest are bound onto the C18 resin in the tip.
6.
Withdraw and
discard to waste 10 µL of 0.1 % TFA five times. This washes away unwanted salts
and contaminants. Make sure you expel all the liquid on the last wash.
7. Withdraw 1 µL of matrix solution. This contains 70% acetonitrile so it is used to elute peptides in the presence of
matrix. (4 mg / mL CHCA
[α-cyano-4-hydroxycynnamic acid], 70 % MeCN, 0.1
% TFA).
8. Spot the eluted peptide sample in
matrix solution directly onto the MALDI target plate in an odd row, trying not
to touch the surface of the plate with the tip. Record the location of your
samples.
9. New C18 tip is needed for
each sample.
10. BSA digest at 1pmol/µL is used as control. This does not required ziptipping.
11. Next, prepare calibration
standards (Pepmix and Matrix solutions at 1:1 ratio),
and spot 1ul of this onto spots in even row next to your samples. These are
your near-point calibration standards.
12.Allow the plate to
air-dry.
13. Plates can be stored in 4oC
fridge and in the dark for up to a week, ready for analysis.
14. Samples are analyzed using the 4800 Plus MALDI Tof-Tof
Analyzer.
LB AGAR PLATE PREPARATION
1. Add 15g Bacto agar to 1000mL
of LB media and autoclave (121oC for 15mins).
2.
Add 1000uL of Chloroamphenicol (25mg/mL), Ampicillin (50mg/mL) or Kanamycin (30mg/mL) and mix well before plating out and
setting the agar.
LB MEDIA
● Ingredients: Tryptone 10g, Yeast extract
5g, NaCl 10g, Milli-Q water
to 1000mL.
● Methods: Dissolved 10g tryptone, 5g
yeast extract and 10g NaCl in 800mL Milli-Q water, making use of a magnetic stirrer. Once
dissolved, bring volume up to 1 L using Milli-Q
water. Autoclave 1L of the solution (121oC, 15 min, standard liquid
cycle).
SOC MEDIA (FOR COMPETENT CELLS)
● Ingredients: 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl 5M NaCl,
417µL 3M KCl, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose
& 500mL Milli-Q water.
● Methods: The adjusted quantities were combined in a 1 L
measuring column with constant stirring and then placed in the autoclave for
sterilisation (121oC, 15 min, standard liquid cycle).
NANODROP for THERMO SCIENTIFIC NanoDrop 2000 Spectrophotometer
1. Pipette 1-2µl of blanking buffer onto the bottom
pedestal, lower the arm and click the BLANK button. The blank solution is the
same buffer that the DNA of interest is dissolved in.
2.
Wipe away the
blank. Pipette 1-2µl of DNA sample and hit MEASURE
3.
Wipe away the
last sample to prepare for the next sample, and repeat.
PCR Mixture: General recipe for PCR is as follows:
● 4 µL of 5x phusion buffer.
● 0.4 µL dNTPs.
● 0.6 µL of DMSO.
● 0.2 µL of polymerase.
● 11.8 µL of water.
To this mixture, add:
● 1 µL of forward primer.
● 1 µL of reverse primer.
● 1 µL of template.
● Total volume = 20 µL
PCR settings: A general program for PCR is as follows:
● Initial denaturation at 98oC
for 30seconds.
Followed by 30 repeats of:
● Denaturation – 98oC for 10seconds.
● Annealing – 60oC for 10seconds.
● Extension – 72oC for 2minutes.
● Final extension – 72oC for 10minutes.
Plasmid Prep
1.
Centrifuge
@13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorf’s
2.
Discard
supernatant and add 1.9 mL of culture and centrifuge
again @13,200 rpm for 10 min
3.
Re-suspend pelleted cells in P1 Buffer (250µl)
4.
Add 250µL of P2
buffer & invert 4-6 times (turns homogenous blue)
5.
Add 350µL of N3
& mix by inverting (turns colourless)
6.
Centrifuge @
10min 13,000 rpm to obtain a pellet
7.
Transfer
supernatant in QIA Prep Spin Column by pipetting
8.
Centrifuge 30-60
sec - discard flow through
9.
Wash QIA Prep
Spin Column with 0.5mL of PB
10.
Centrifuge for
30-60 seconds - discard flow through
11.
Wash spin column
by adding 0.75 mL PE buffer
12.
Centrifuge for
30-60 seconds - discard flow through and centrifuge for a further 1min
13.
Place QIAPrep Column in clean Eppendorf
14.
Elute DNA add
50µl of water, stand for 1min, centrifuge for 1min
15.
QIA prep - Spin miniprep buffer
SDS-PAGE
1.
Re-suspend pelleted bacterial cells in 200µL of Milli-Q-water
2.
Transfer 50 µL of
suspension into new Eppendorf tubes and combine with
50ul of 2xTruSep sample buffer.
3.
Shear the cells using a Hamilton
syringe.
4.
Centrifuge the
preparation for 3minutes @ 13,000 rpm.
5.
Load 20 µL of the
supernatant into gel.
6.
Conduct
electrophoresis at a constant voltage (200V) for 1 hour.
7.
Coommassie Stain for ~30 minutes.
Heat Shock
1.
Obtain competent
cells from -80oC.
2.
Defrost gently on
ice. 100µl is sufficient for 2 transformations.
3.
Add 1-10µl of
plasmid DNA/ ligation mix to each tube. Incubate on ice for 5 min.
4.
Put the tubes in
the 42oC water bath for 30 seconds, then
back on ice for 2 min.
5.
Add 200µl of SOC
media to each tube, and incubate in the 37oC shaker for 10 min or up
to 1h (10min for purified plasmid or 1h for ligation mix).
6.
For each tube of
cells, spread 50µl onto one LB plate with appropriate antibiotic, and 500µl
onto a second plate, using aseptic technique. Place your plate upside-down in
the 37oC incubator.
Induction of Operons
1. Re-transform restriction-digest screened plasmids
according to the heat shock transformation protocol.
2.
Screen plates for
transformant colonies and place in 5mL of ZYM-5052 broth with appropriate
antibiotic concentration at 37oC with shaking until OD600¬ is 2.0
– 3.0.
3.
Take whole volume
and place in 45mL of ZYM-5052 broth in a sterile conical flask containing
appropriate antibiotic concentration. Incubate at 37oC with shaking.
4.
Cell pellets were
collected through centrifugation at 12000 rpm for 5 mins.
5.
Cell pellets were
resuspended in re-suspension buffer (Containing: 10% glycerol w/v, 50mM Tricene NaOH pH 8.0, 2mM MgCl2,
1mM DTT) and whole lysate was collected through a French Press for functional
assays of operon protein products in lysate.
MgPE enzymatic functional assay
1. Collect whole lysate, from induced operon 3 containing ChlM
expressed proteins, using ChlM pET
(L. Meinecke et al.2010)as a
negative control.
2.
Prepare a master
mix of recombinant ChlM, 25µl of S-Adenosyl-L- methionine and 65µl
of 2µM of Mg-protoporphyrin IX.
3.
Incubate at 30℃ with gentle
agitation for 8-10min
4.
100µL of acetone
was added to precipitate protein.
5.
The sample was
spun down, to separate the protein and supernatant.
6.
Supernatant was
then subjected to UHPLC to and observed peaks.
UHPLC protocol
The UHPLC protocol was performed
according to Sawicki and Willows (2007). Briefly, a
Shimadzu HPLC system was used at 2ml/min with an Alltech
C8 column. A 5 min linear gradient was used for separation of Mg-protoporphyrin IX and Magnesium-protoporphyrin
IX mono-methyl ester. Compounds were detected at an excitation wavelength of
excitation of 416nm and emission of 595nm.
Auto induction recipe
1.ZYM-5052 Medium ● ZY media 9.57ml ● 1m MgSO4 20ul ● 50x5052 200ul ● 50 x M 200ul |
2. 50 x M ● DI water 80ml ● Na2HPO4 17.75g ● KH2PO4 17g ● NH4Cl 13.4 ● Na2SO4 3.55g |
3. 50 x 5052 ● Glycerol 25g (weigh in Beaker) ● DI water 73ml ● Glucose 2.5g ● alpha lactose 10g |
4. 1M MgSO4 ● MgSO4.7H2O 24.65g ● DI water 87ml ● 5. ZY Media (400mL) ● Trypton 4g ● Yeast extract 2g |
References
● sawicki,A.,Willows,R.D.(2007).S-Adenosyl-L-methionine:magnesium-protoporphyrin
IX O-methyltransferase from Rhodebacter
capsulatus: mechanistic insights and stimulation with
phospholipids. Journal of Biochemistry, 406, 469-478. doi: 10.1042/BJ20070284
● William,F., Studier. (2014). Stable Expression
Clones and Auto-Induction for Protein Production in E.coli. Structural Genomics: General Applications, Methods in
Molecular Biology.1091, 17-32 DOI 10.1007/978-1-62703-691-7_2
● Meinecke, L., Alawady, A., Schroda, M., Willows, R., Kobayashi, M.C,.
Niyogi, K.K,. Grimm, B.,
Beck, C.F. (2010). Chlorophyll-deficient mutants of Chlamydomonas
reinhardtii that accumulate magnesium protoporphyrin IX. doi:
10.1007/s11103-010-9604-9