|
|
| Line 9: |
Line 9: |
| | <details> | | <details> |
| | <summary>7/19/2015 - 7/25/2015</summary> | | <summary>7/19/2015 - 7/25/2015</summary> |
| | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/21_July_2015"><li>7/21/2015 |
| | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/20_July_2015"><li>7/20/2015 - Cloning M2-9(T7) and M2-12(1C3), MaSp1 PCR End-Extension</li></a> | | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/20_July_2015"><li>7/20/2015 - Cloning M2-9(T7) and M2-12(1C3), MaSp1 PCR End-Extension</li></a> |
| | </details> | | </details> |
| Line 43: |
Line 44: |
| | <summary>6/14/2015 - 6/20/2015</summary> | | <summary>6/14/2015 - 6/20/2015</summary> |
| | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/19_June_2015"><li>6/19/2015 - Initial ICA Testing</li></a> | | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/19_June_2015"><li>6/19/2015 - Initial ICA Testing</li></a> |
| − | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/18_June_2015"><li>6/18/2015 - BsaI Digestion of VF/R Amplified MaSp</li></a> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/18_June_2015"><li>6/18/2015 - BsaI Digestion of VF/R Amplified MaSp2</li></a> |
| | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/17_June_2015"><li>6/17/2015 - PCR amplification of MaSp using VF2, VR</li></a> | | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/17_June_2015"><li>6/17/2015 - PCR amplification of MaSp using VF2, VR</li></a> |
| | </details> | | </details> |
| Line 50: |
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| | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/5_June_2015"><li>6/5/2015 - ICA Oligos Characterization</li></a> | | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/5_June_2015"><li>6/5/2015 - ICA Oligos Characterization</li></a> |
| | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/3_June_2015"><li>6/3/2015 - ICA Oligos Characterization</li></a> | | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/3_June_2015"><li>6/3/2015 - ICA Oligos Characterization</li></a> |
| − | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/2_June_2015"><li>6/2/2015 - Gel purification of BsaI digested MaSp</li></a> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/2_June_2015"><li>6/2/2015 - Gel purification of BsaI digested MaSp2</li></a> |
| | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/1_June_2015"><li>6/1/2015 - Preparing to test Initiator, Terminator, and Capping Oligos: PCR amplification and BsaI Digestion</li></a> | | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/1_June_2015"><li>6/1/2015 - Preparing to test Initiator, Terminator, and Capping Oligos: PCR amplification and BsaI Digestion</li></a> |
| | </details> | | </details> |
| Line 56: |
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| | <summary>5/24/2015 - 5/30/2015</summary> | | <summary>5/24/2015 - 5/30/2015</summary> |
| | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/29_May_2015"><li>5/29/2015 - T7 Ligation Test for VF/R amplified MaSp</li></a> | | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/29_May_2015"><li>5/29/2015 - T7 Ligation Test for VF/R amplified MaSp</li></a> |
| − | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/28_May_2015"><li>5/28/2015 - BsaI Digestion of VF/R amplified MaSp</li></a> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/28_May_2015"><li>5/28/2015 - BsaI Digestion of VF/R amplified MaSp2</li></a> |
| | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/27_May_2015"><li>5/27/2015 - Temperature Test for VF/R amplification of MaSp, Large-scale amplification of MaSp 2AB, 2BC, 2CA</li></a> | | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/27_May_2015"><li>5/27/2015 - Temperature Test for VF/R amplification of MaSp, Large-scale amplification of MaSp 2AB, 2BC, 2CA</li></a> |
| − | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/26_May_2015"><li>5/26/2015 - BsaI Digestion of VF/R amplified MaSp</li></a> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/26_May_2015"><li>5/26/2015 - BsaI Digestion of VF/R amplified MaSp2</li></a> |
| | </details> | | </details> |
| | <details> | | <details> |
| | <summary>5/17/2015 - 5/23/2015</summary> | | <summary>5/17/2015 - 5/23/2015</summary> |
| − | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/22_May_2015"><li>5/22/2015 - PCR amplification of MaSp using VF2, VR</li></a> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/22_May_2015"><li>5/22/2015 - PCR amplification of MaSp2 using VF2, VR</li></a> |
| − | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/21_May_2015"><li>5/21/2015 - PCR amplification of MaSp using VF2, VR Cycle Testing</li></a> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/21_May_2015"><li>5/21/2015 - PCR amplification of MaSp2 using VF2, VR Cycle Testing</li></a> |
| − | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/20_May_2015"><li>5/20/2015 - PCR amplification of MaSp using VF2, VR</li></a> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/20_May_2015"><li>5/20/2015 - PCR amplification of MaSp2 using VF2, VR</li></a> |
| − | <details> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/19_May_2015"><li>5/19/2015 - PCR amplification test for MaSp2 plasmid</li></a> |
| − | <summary>5/19/2015 - PCR amplification test for MaSp plasmid</summary>
| + | |
| − | <head><title>5/19/2015</title>
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| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
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| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
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| − | <!-- html --> | + | |
| − | <meta name="src" content="5_19_2015.tex">
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| − | <meta name="date" content="2015-05-19 22:26:00">
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| − | <link rel="stylesheet" type="text/css" href="5_19_2015.css">
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| − | </head><body
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| − | >
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| − | <div class="maketitle">
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| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">5/19/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>PCR Amplification of MaSp Plasmids</h3>
| + | |
| − | <!--l. 14--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Set up four 25 uL PCR reactions to test amplification. Used two pairs of
| + | |
| − | primers and two different cycling conditions.
| + | |
| − | <!--l. 16--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Used MaSp2 2CA as template. Diluted 1:1000 to get 219 pg/uL. Created 2
| + | |
| − | master-mixes, one with each primer pair.
| + | |
| − | <!--l. 18--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-40001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Tested VF2/VR primers and post-elution primers using 17 cycles and 15
| + | |
| − | cycles using protocol below: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 20--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">1x Reaction (uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2x Reaction (uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">5x Q5 Buffer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">10 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">10 mM dNTPs </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">0.5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">1 </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">10 uM For </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">1.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-3"
| + | |
| − | class="td11">2.5</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">10 uM Rev </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">1.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-3"
| + | |
| − | class="td11">2.5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11">Template (110 pg)</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11">0.5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-3"
| + | |
| − | class="td11">1 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11">5x GC Buffer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11">5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-3"
| + | |
| − | class="td11">10 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-1"
| + | |
| − | class="td11">Q5 Polymerase </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-2"
| + | |
| − | class="td11">0.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-3"
| + | |
| − | class="td11">0.5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-2"
| + | |
| − | class="td11">11.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-3"
| + | |
| − | class="td11">22.5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-10-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">25 </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 </span></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 34--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11">30 sec </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">10 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-1"
| + | |
| − | class="td11">66 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-2"
| + | |
| − | class="td11">15 sec </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-2"
| + | |
| − | class="td11">15 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-1"
| + | |
| − | class="td11">repeat from step 2</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-2"
| + | |
| − | class="td11">17x/15x</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-2"
| + | |
| − | class="td11">2 min </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-1"
| + | |
| − | class="td11">12 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-2"
| + | |
| − | class="td11">hold </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 45--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-50001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | We used 66 C as annealing because that temperature worked for previous
| + | |
| − | amplification with the post-elution primers. In addition, NEB Tm calculator
| + | |
| − | indicates 66 C annealing for the VF2/VR primer pair.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-60002"></a>Results</h3>
| + | |
| − | <!--l. 48--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-70002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Cast 2% TAE gel to visualize results. Used 2 uL of NEB 50 bp ladder.
| + | |
| − | <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-7001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 52--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/d/d3/5_19_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">P/E indicates reactions with post-elution primer pair. VF/R
| + | |
| − | indicates reactions with VF2 and VR primers. The VF/R primers successfully
| + | |
| − | amplify with a clean band. P/E does not appear to amplify. Primer dimers
| + | |
| − | are present in all lanes. Imaged by Danny. Don’t know why the image is
| + | |
| − | orange.</span></div><!--tex4ht:label?: x1-7001r1 -->
| + | |
| − | | + | |
| − | <!--l. 54--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 55--><p class="indent" > <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-7002r2"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 57--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/7/72/5_19_2015_2.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 2: </span><span
| + | |
| − | class="content">P/E indicates reactions with post-elution primer pair. VF/R
| + | |
| − | indicates reactions with VF2 and VR primers. The VF/R primers successfully
| + | |
| − | amplify with a clean band. P/E does not appear to amplify. Primer dimers
| + | |
| − | are present in all lanes. This image taken several hours after previous image.
| + | |
| − | Some band smearing is present.</span></div><!--tex4ht:label?: x1-7002r2 -->
| + | |
| − | | + | |
| − | <!--l. 59--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 60--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-80002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | The VF/R primer pair amplifies our plasmid as expected, but the
| + | |
| − | post-elution primers do not. The expected size for VF/R amplification is 443
| + | |
| − | bp.
| + | |
| − | <!--l. 62--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-90002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | May be due to bad primers for post-elution. Re-order or remake? We need to
| + | |
| − | find more of the post-elution primers.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| | </details> | | </details> |
| − |
| |
| | <details> | | <details> |
| | <summary>5/10/2015 - 5/16/2015</summary> | | <summary>5/10/2015 - 5/16/2015</summary> |
| − | <details> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/13_May_2015"><li>5/13/2015 - NotI Concatemer Ligation</li></a> |
| − | <summary>5/13/2015 - NotI Concatemer Ligation</summary>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/12_May_2015"><li>5/12/2015 - T4/T7 Ligation Tests continued</li></a> |
| − | <head><title>5/13/2015</title>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/11_May_2015"><li>5/11/2015 - BsaI Digestion of MaSp2 Plasmids</li></a> |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
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| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html --> | + | |
| − | <meta name="src" content="5_13_2015.tex">
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| − | <meta name="date" content="2015-05-15 15:14:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="5_13_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">5/13/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>NotI Concatemers</h3>
| + | |
| − | <!--l. 14--><p class="noindent" >Used yesterday’s gel purified fragments. We performed ligation using a 5:1 ratio of
| + | |
| − | insert to vector. Our vector size is 2046 bp. Our insert is 143 bp. For 1121 ng of
| + | |
| − | vector, we need 391.75 ng of insert. We set up the ligation reaction as below:
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 15--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11">Amount (uL)</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">Vector (112.12 ng/uL)</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">10 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">Insert (52.63 ng/uL) </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">10 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">10X T4 Ligase Buffer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">2.5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">T4 Ligase </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">3 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">25.5 </span></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" /> | + | |
| − | </div>
| + | |
| − | <!--l. 26--><p class="indent" > We used excess insert because the recorded concentration from the nanodrop
| + | |
| − | was higher than the theoretical yield. In addition, we used more T4 Ligase than in
| + | |
| − | typical reactions due to the reaction having three times as many sticky ends to
| + | |
| − | ligate.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a | + | |
| − | id="x1-20002"></a>Results</h3>
| + | |
| − | <!--l. 28--><p class="noindent" >We cast a 0.8% TAE agarose gel to separate the bands. We ran this particular gel
| + | |
| − | at 90 V for 2 hours. We excised four bands indicated for gel purification (Qiagen).
| + | |
| − | The bottom most band may be due to vector self-ligation. The second band at 2.2
| + | |
| − | kb is probably singly ligated product. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-2001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 31--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/thumb/a/a1/5_13_2015.jpg/424px-5_13_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Ligation of 2CA NotI digested vector and insert. We excised the
| + | |
| − | 4 indicated bands for purification. </span></div><!--tex4ht:label?: x1-2001r1 -->
| + | |
| − | | + | |
| − | <!--l. 33--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 34--><p class="indent" > In the future, we should CIP treat the vector so it doesn’t re-ligate.
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>5/12/2015 - T4/T7 Ligation Tests continued</summary>
| + | |
| − | <head><title>5/12/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="5_12_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-13 18:35:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="5_12_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">5/12/2015</h2>
| + | |
| − | <div class="author" ><span
| + | |
| − | class="cmr-12x-x-120">Vinson Lam</span></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Gel Extraction</h3>
| + | |
| − | <!--l. 14--><p class="noindent" >Performed gel extraction (Qiagen kit) of bands excised from yesterday’s gel.
| + | |
| − | Results below: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 15--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"><col
| + | |
| − | id="TBL-2-4"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Concentration (ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">A 260/280</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">2AB </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">41.5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">4.47 </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">2BC </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">54.3 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">3.96</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">2CA </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">17.6 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-3"
| + | |
| − | class="td11">3.21 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">2CA NotI ins.</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">52.63 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-3"
| + | |
| − | class="td11">3.70 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11">2CA NotI vec.</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11">112.12 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-3"
| + | |
| − | class="td11">2.50 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11"> </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" /> | + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a | + | |
| − | id="x1-20002"></a>2AB, 2BC, 2CA BsaI Ligation Test</h3>
| + | |
| − | <!--l. 27--><p class="noindent" >Set up ligation tests for remaining conditions (see entry for 5/8/2015)for other
| + | |
| − | tests. For each reaction, we used 50 ng of each DNA species.
| + | |
| − | <h4 class="subsectionHead"><span class="titlemark">2.1 </span> <a
| + | |
| − | id="x1-30002.1"></a>T4 Ligase Reactions</h4>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 29--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"><col
| + | |
| − | id="TBL-3-3"><col
| + | |
| − | id="TBL-3-4"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2AB + 2BC</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2BC +2CA</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">10X T4 Ligase Buffer</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">1.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-3"
| + | |
| − | class="td11">1.5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-1"
| + | |
| − | class="td11">2AB (6.82 ng/uL) </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-2"
| + | |
| − | class="td11">7.3 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-3"
| + | |
| − | class="td11">0 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-1"
| + | |
| − | class="td11">2BC (54.3 ng/uL) </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-2"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-3"
| + | |
| − | class="td11">1 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-1"
| + | |
| − | class="td11">2CA (17.6 ng/uL) </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-2"
| + | |
| − | class="td11">0 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-3"
| + | |
| − | class="td11">2.84 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-1"
| + | |
| − | class="td11">T4 Ligase </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-2"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-3"
| + | |
| − | class="td11">1 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-2"
| + | |
| − | class="td11">4.2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-3"
| + | |
| − | class="td11">8.66 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">15 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">15 uL </span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-9-1"
| + | |
| − | class="td11"> </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 42--><p class="indent" > Reactions were incubated at 25 C for 10 min.
| + | |
| − | <h4 class="subsectionHead"><span class="titlemark">2.2 </span> <a
| + | |
| − | id="x1-40002.2"></a>T7 Ligase Reactions</h4>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 44--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-4" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-4-1g"><col
| + | |
| − | id="TBL-4-1"><col
| + | |
| − | id="TBL-4-2"><col
| + | |
| − | id="TBL-4-3"><col
| + | |
| − | id="TBL-4-4"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2CA + 2AB</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2BC + 2CA</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-1"
| + | |
| − | class="td11">2X T7 Ligase Buffer</td><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-2"
| + | |
| − | class="td11">7.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-3"
| + | |
| − | class="td11">7.5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-1"
| + | |
| − | class="td11">2AB </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-2"
| + | |
| − | class="td11">1.2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-3"
| + | |
| − | class="td11">0 uL </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-4-1"
| + | |
| − | class="td11">2BC </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-4-2"
| + | |
| − | class="td11">0 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-4-3"
| + | |
| − | class="td11">1 uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-5-1"
| + | |
| − | class="td11">2CA </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-5-2"
| + | |
| − | class="td11">2.84 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-5-3"
| + | |
| − | class="td11">2.84 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-6-1"
| + | |
| − | class="td11">T7 Ligase </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-6-2"
| + | |
| − | class="td11">0.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-6-3"
| + | |
| − | class="td11">0.5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-7-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-7-2"
| + | |
| − | class="td11">2.96 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-7-3"
| + | |
| − | class="td11">3.16 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-8-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-4-8-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">15 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-4-8-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">15 uL </span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-9-1"
| + | |
| − | class="td11"> </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 57--><p class="indent" > Reactions were incubated at 25 C for 10 min.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">3 </span> <a
| + | |
| − | id="x1-50003"></a>Results</h3>
| + | |
| − | <!--l. 59--><p class="noindent" >We cast a 2% TAE agarose gel to visualize the results. We used 2 uL of 50 bp
| + | |
| − | ladder. See below. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-5001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 62--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/thumb/8/86/Ligation_Test_T4_T7.jpg/722px-Ligation_Test_T4_T7.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">T4 T7 Ligation Test</span></div><!--tex4ht:label?: x1-5001r1 -->
| + | |
| − | | + | |
| − | <!--l. 64--><p class="indent" > </div><hr class="endfigure">
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>5/11/2015 - BsaI Digestion of MaSp Plasmids</summary>
| + | |
| − | <head><title>5/11/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="5_11_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-11 23:16:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="5_11_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">5/11/2015</h2>
| + | |
| − | <div class="author" ><span
| + | |
| − | class="cmr-12x-x-120">Vinson Lam</span></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>2AB, 2BC, 2CA Ligation Test</h3>
| + | |
| − | <!--l. 14--><p class="noindent" >
| + | |
| − | <h4 class="subsectionHead"><span class="titlemark">1.1 </span> <a
| + | |
| − | id="x1-20001.1"></a>BsaI digestion of 2AB, 2BC, 2CA</h4>
| + | |
| − | <!--l. 15--><p class="noindent" >Used 5 ug of each DNA. <div class="table">
| + | |
| − | | + | |
| − | <!--l. 16--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"><col
| + | |
| − | id="TBL-2-4"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11">2AB (112 ng/uL)</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11">2BC (205 ng/uL)</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-4"
| + | |
| − | class="td11">2CA (218.7 ng/uL)</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">BsaI </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">4 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">4 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-4"
| + | |
| − | class="td11">4 uL </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">Template</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">44.6 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">24.39 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-4"
| + | |
| − | class="td11">22.86 uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">Cutsmart</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-3"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-4"
| + | |
| − | class="td11">5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">0 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-3"
| + | |
| − | class="td11">16.61 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-4"
| + | |
| − | class="td11">18.14 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">53.6 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL </span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-3"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-4"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-1"
| + | |
| − | class="td11">50 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-2"
| + | |
| − | class="td11">2 hr </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-3"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-4"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-1"
| + | |
| − | class="td11">65 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-2"
| + | |
| − | class="td11">20 min </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-3"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-4"
| + | |
| − | class="td11"> </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 29--><p class="indent" > We are conducting ligation tests between all possible combinations using T4
| + | |
| − | and T7 ligase
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-30002"></a>NotI Concatemers</h3>
| + | |
| − | <!--l. 31--><p class="noindent" >Sri suggested using NotI to digest the plasmids and create a super plasmid that
| + | |
| − | contains multiple copies of the monomers. <br
| + | |
| − | class="newline" />Testing NotI digestion today using 2CA with the protocol below. <div class="table">
| + | |
| − | | + | |
| − | <!--l. 33--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11">2CA (280 ng/uL)</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">NotI-HF </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">2 uL </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-1"
| + | |
| − | class="td11">DNA </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-2"
| + | |
| − | class="td11">7.2 uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-1"
| + | |
| − | class="td11">Cutsmart</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-2"
| + | |
| − | class="td11">5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-2"
| + | |
| − | class="td11">35.8 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL </span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-2"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-1"
| + | |
| − | class="td11">37 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-2"
| + | |
| − | class="td11">1 hr </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-9-1"
| + | |
| − | class="td11">65 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-9-2"
| + | |
| − | class="td11">20 min </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">3 </span> <a
| + | |
| − | id="x1-40003"></a>Results</h3>
| + | |
| − | <!--l. 47--><p class="noindent" >Cast 200 mL of a 1.5% TAE agarose gel to visualize results of both BsaI and Not1
| + | |
| − | digestion. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-4001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 50--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/7/7a/BsaI_NotI.jpg" width="500"alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Digestion Results: The 100 bp bands were excised as well as the
| + | |
| − | second band in the NotI digestion.</span></div><!--tex4ht:label?: x1-4001r1 -->
| + | |
| − | | + | |
| − | <!--l. 52--><p class="indent" > </div><hr class="endfigure">
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| | </details> | | </details> |
| − |
| |
| | <details> | | <details> |
| | <summary>5/3/2015 - 5/9/2015</summary> | | <summary>5/3/2015 - 5/9/2015</summary> |
| − | <details> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/8_May_2015"><li>5/8/2015 - T4/T7 Ligation Test</li></a> |
| − | <summary>5/8/2015 - T4/T7 Ligation Test</summary>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/7_May_2015"><li>5/7/2015 - BsaI Digestion of MaSp plasmids</li></a> |
| − | <head><title>5/8/2015</title>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/6_May_2015"><li>5/6/2015 - DNA Maxiprep</li></a> |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/5_May_2015"><li>5/5/2015 - DNA Miniprep</li></a> |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/4_May_2015"><li>5/4/2015 - BsaI Digestion Test</li></a> |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html --> | + | |
| − | <meta name="src" content="5_8_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-14 20:17:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="5_8_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">5/8/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>T4/T7 Ligation Test</h3>
| + | |
| − | <!--l. 14--><p class="noindent" >We tested the ability for the BsaI digested and gel purified monomers to ligate
| + | |
| − | together. We ran these initial tests using both T4 and T7 DNA ligase using the
| + | |
| − | following protocols (50 ng for each DNA species):
| + | |
| − | <h4 class="subsectionHead"><span class="titlemark">1.1 </span> <a
| + | |
| − | id="x1-20001.1"></a>T4 Ligation</h4>
| + | |
| − | <div class="table"> | + | |
| − | | + | |
| − | <!--l. 16--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11">10x T4 Ligase Buffer</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11">1.5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">2AB </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">4 uL </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">2CA </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">5 uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">T4 Ligase </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">1 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">3.5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">15 uL</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-3"
| + | |
| − | class="td11"></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-1"
| + | |
| − | class="td11">25 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-2"
| + | |
| − | class="td11">10 min</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-1"
| + | |
| − | class="td11"> </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h4 class="subsectionHead"><span class="titlemark">1.2 </span> <a | + | |
| − | id="x1-30001.2"></a>T7 Ligation</h4>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 30--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"><col
| + | |
| − | id="TBL-3-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11">2x T7 Ligase Buffer</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11">7.5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">2AB </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">4 uL </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-1"
| + | |
| − | class="td11">2BC </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-2"
| + | |
| − | class="td11">5 uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-1"
| + | |
| − | class="td11">T7 Ligase </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-2"
| + | |
| − | class="td11">0.5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-2"
| + | |
| − | class="td11">0 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">17 uL</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-2"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-3"
| + | |
| − | class="td11"></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-1"
| + | |
| − | class="td11">25 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-2"
| + | |
| − | class="td11">10 min</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-9-1"
| + | |
| − | class="td11"> </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-40002"></a>Results</h3>
| + | |
| − | <!--l. 44--><p class="noindent" >We cast a 2% TAE gel to visualize the results. We use 2 uL of NEB 50 bp ladder.
| + | |
| − | The gel was run at 150V for 35 min. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-4001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 47--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/a/ac/5_8_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Preliminary ligation test with T4 and T7 polymerase. The band
| + | |
| − | slightly larger than 200 bp is our desired ligation product.</span></div><!--tex4ht:label?: x1-4001r1 -->
| + | |
| − | | + | |
| − | <!--l. 49--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 50--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-50002"></a></span>
| + | |
| − | Our ligation test indicates that the new sticky ends exhibit specific binding. In
| + | |
| − | both reactions, the ligation was incomplete due to the presence of a band at 100
| + | |
| − | bp. This may be due to non-equal amounts of DNA.
| + | |
| − | <!--l. 52--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-60002"></a></span>
| + | |
| − | We plan to conduct this same test next week using all possible combinations
| + | |
| − | between the three monomers to verify specific binding. We plan to test both T4
| + | |
| − | and T7.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>5/7/2015 - BsaI Digestion of MaSp plasmids</summary>
| + | |
| − | <head><title>5/7/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1"> | + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html --> | + | |
| − | <meta name="src" content="5_7_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-14 20:37:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="5_7_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">5/7/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>BsaI Digestion of 2AB, 2BC, 2CA</h3>
| + | |
| − | <!--l. 14--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Gel purification of yesterday’s BsaI digested fragments (Qiagen).
| + | |
| − | <!--l. 16--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Obtained approx. 7 ng/uL for each monomer. A 260/280 for all three were
| + | |
| − | around 1.8. Will use these as is for ligation test tomorrow.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>5/6/2015 - DNA Maxiprep</summary>
| + | |
| − | | + | |
| − | <head><title>5/6/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
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| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="5_6_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-16 10:10:00">
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| − | <link rel="stylesheet" type="text/css" href="5_6_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">5/6/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <!--l. 13--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-1000"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Spun down yesterday’s cells in 50 mL tubes for 15 min @ 6000x g @ 4 | + | |
| − | C.
| + | |
| − | <!--l. 15--><p class="noindent" ><span class="paragraphHead"><a | + | |
| − | id="x1-2000"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Used Maxiprep (Qiagen) kit to prepare DNA. Eluted in 400 uL EB.
| + | |
| − | Concentrations from each are listed below: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 17--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11">Amount (ng/uL)</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">2AB</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">112 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">2BC</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">205 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">2CA</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">219 </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 26--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-3000"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | The DNA amounts are low. We probably need to improve our technique.
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>5/5/2015 - DNA Miniprep</summary>
| + | |
| − | <head><title>5/5/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="5_5_2015.tex">
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| − | <meta name="date" content="2015-05-16 11:39:00">
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| − | <link rel="stylesheet" type="text/css" href="5_5_2015.css">
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| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">5/5/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <!--l. 12--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-1000"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Miniprep (Zymo) yesterday’s overnight bacterial culture. We also created | + | |
| − | glycerol stocks from these cultures. <div class="table">
| + | |
| − | | + | |
| − | <!--l. 14--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11">Amount (ng/uL)</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">2AB</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">380 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">2BC</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">460 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">2CA</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">219 </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 22--><p class="noindent" ><span class="paragraphHead"><a | + | |
| − | id="x1-2000"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Prepared 100 mL culture of 2AB, 2BC, 2CA for Maxiprep tomorrow.
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>5/4/2015 - BsaI Digestion Tes</summary>
| + | |
| − | <head><title>5/4/2015</title>
| + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
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| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
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| − | <meta name="date" content="2015-05-16 21:20:00">
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| − | <div class="maketitle">
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| − | <h2 class="titleHead">5/4/2015</h2>
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| − | <div class="author" ></div><br />
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| − | <div class="date" ></div>
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| − | </div>
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| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
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| − | id="x1-10001"></a>BsaI Digestion Test for 2AB</h3>
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| − | <!--l. 14--><p class="noindent" >Tested digestion of 5 ug of plasmid to see efficiency of digestion. <div class="table">
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| − | | + | |
| − | <!--l. 15--><p class="indent" > <hr class="float"><div class="float"
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| − | >
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| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
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| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
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| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
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| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11">Amount (uL)</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">BsaI </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
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| − | class="td11">2 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
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| − | class="td11">2AB (287 ng/uL)</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
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| − | class="td11">14.7 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">Cutsmart </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">28.28 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
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| − | class="td11"><span
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| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL </span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
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| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
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| − | style="vertical-align:baseline;" id="TBL-2-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-1"
| + | |
| − | class="td11">50 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-2"
| + | |
| − | class="td11">2 hr </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-1"
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| − | class="td11">65 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-2"
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| − | class="td11">20 min </td></tr></table></div>
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| − | | + | |
| − | </div><hr class="endfloat" />
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| − | </div>
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| − | <!--l. 29--><p class="indent" > Results are shown below: <hr class="figure"><div class="figure"
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| − | >
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| − | | + | |
| − | <a
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| − | id="x1-1001r1"></a>
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| − | | + | |
| − | | + | |
| − | <!--l. 32--><p class="noindent" ><img
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| − | src="https://static.igem.org/mediawiki/2015/1/18/5_4_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Results of BsaI digestion test. The band at about 100 bp
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| − | corresponds to the target digested insert.</span></div><!--tex4ht:label?: x1-1001r1 -->
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| − | | + | |
| − | <!--l. 34--><p class="indent" > </div><hr class="endfigure">
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| − | <!--l. 35--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | The digestion process works, so we’ll be proceeding with this protocol.
| + | |
| − | <!--l. 37--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | We gel extracted the digested insert band. The end result was 11.9 ng/uL, A
| + | |
| − | (260/280): 4.4
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-40002"></a>BsaI Digestion for Post-elution primer amplified MaSp</h3>
| + | |
| − | <!--l. 40--><p class="noindent" >We BsaI digested the PCR products from previous PCRs using the following
| + | |
| − | protocol
| + | |
| − | <h4 class="subsectionHead"><span class="titlemark">2.1 </span> <a
| + | |
| − | id="x1-50002.1"></a>BsaI Digest Protocol</h4>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 42--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"><col
| + | |
| − | id="TBL-3-3"><col
| + | |
| − | id="TBL-3-4"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2AB(61.43 ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2BC* </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">CA (56.65 ng/uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">BsaI </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-3"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-4"
| + | |
| − | class="td11">2 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-1"
| + | |
| − | class="td11">DNA (500 ng)</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-2"
| + | |
| − | class="td11">8.14 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-3"
| + | |
| − | class="td11">18.01 uL</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-4"
| + | |
| − | class="td11">8.83 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-1"
| + | |
| − | class="td11">10x Cutsmart </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-2"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-3"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-4"
| + | |
| − | class="td11">5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-2"
| + | |
| − | class="td11">34.86 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-3"
| + | |
| − | class="td11">25 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-4"
| + | |
| − | class="td11">34.17 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL </span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-2"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-3"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-4"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-1"
| + | |
| − | class="td11">50 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-2"
| + | |
| − | class="td11">2 hr </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-3"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-8-4"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-9-1"
| + | |
| − | class="td11">65 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-9-2"
| + | |
| − | class="td11">20 min </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-9-3"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-9-4"
| + | |
| − | class="td11"> </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 56--><p class="indent" > *For 2BC, we combined 8.84 uL of 33.9 ng/uL solution and 9.17 uL and 33.9
| + | |
| − | ng/uL solution to get 500 ng total.
| + | |
| − | <h4 class="subsectionHead"><span class="titlemark">2.2 </span> <a
| + | |
| − | id="x1-60002.2"></a>Purification</h4>
| + | |
| − | <!--l. 58--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-70002.2"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | PCR purified the post-elution PCR digestion products and got the following
| + | |
| − | yields: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 60--><p class="indent" > <a
| + | |
| − | id="x1-7001r1"></a><hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-4" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-4-1g"><col
| + | |
| − | id="TBL-4-1"><col
| + | |
| − | id="TBL-4-2"><col
| + | |
| − | id="TBL-4-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-2"
| + | |
| − | class="td11">Concentration (ng/uL)</td><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-3"
| + | |
| − | class="td11">A (260/280)</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-1"
| + | |
| − | class="td11">2AB</td><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-2"
| + | |
| − | class="td11">3.74 </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-3"
| + | |
| − | class="td11">1.22 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-1"
| + | |
| − | class="td11">2BC</td><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-2"
| + | |
| − | class="td11">9.81 </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-3"
| + | |
| − | class="td11">2.54 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-4-1"
| + | |
| − | class="td11">2CA</td><td style="white-space:nowrap; text-align:left;" id="TBL-4-4-2"
| + | |
| − | class="td11">11.38 </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-4-3"
| + | |
| − | class="td11">2.33 </td></tr></table></div>
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Table 1: </span><span
| + | |
| − | class="content">PCR purification of BsaI digested fragments generated through
| + | |
| − | PCR using post-elution primers.</span></div><!--tex4ht:label?: x1-7001r1 -->
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 70--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-80002.2"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Yield and absorbance for the digested inserts are not very good. This
| + | |
| − | might be a problem due to the PCR process, even though product was
| + | |
| − | generated.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">3 </span> <a
| + | |
| − | id="x1-90003"></a>Bacterial Propagation</h3>
| + | |
| − | <!--l. 73--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-100003"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Grew 7 mL each of AB, BC, CA from glycerol stock for DNA extraction
| + | |
| − | tomorrow.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| | </details> | | </details> |
| − |
| |
| | <details> | | <details> |
| | <summary>4/26/2015 - 5/2/2015</summary> | | <summary>4/26/2015 - 5/2/2015</summary> |
| − | <details> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/1_May_2015"><li>5/1/2015 - Primer Exhaustion Calculation</li></a> |
| − | <summary>5/1/2015 - Primer Exhaustion Calculation</summary>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/30_April_2015"><li>4/30/2015 - Post-elution PCR amplification of MaSp2 plasmids</li></a> |
| − | <head><title>5/1/2015</title>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/29_April_2015"><li>4/29/2015 - Post-elution PCR amplification of MaSp2 plasmids</li></a> |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/28_April_2015"><li>4/28/2015 - Post-elution PCR amplification of MaSp2 plasmids</li></a> |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/27_April_2015"><li>4/27/2015 - Initial ICA Ligation Test</li></a> |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html --> | + | |
| − | <meta name="src" content="5_1_2015.tex">
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| − | <meta name="date" content="2015-05-18 23:29:00">
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| − | <link rel="stylesheet" type="text/css" href="5_1_2015.css">
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| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">5/1/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Calculation for Primer exhaustion</h3>
| + | |
| − | <!--l. 13--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Vector+insert = 2205 bp (150 bp insert) (2055 bp vector) | + | |
| − | <!--l. 15--><p class="noindent" ><span class="paragraphHead"><a | + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | DNA: 649 g/mole/bp
| + | |
| − | <!--l. 17--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-40001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | AB: 143105 g/mole
| + | |
| − | <!--l. 19--><p class="indent" > <span class="subparagraphHead"> <a
| + | |
| − | id="x1-50001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span> 500 pg / 1431045 g/mol = 3.494 E -16 moles DNA
| + | |
| − | <!--l. 21--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-60001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | 1.25 uL of 10 uM primers = 1.25 E -11 moles primers
| + | |
| − | | + | |
| − | <!--l. 23--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-70001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | 1.25 E -11 / 3.494 E -16 = 35775.6
| + | |
| − | <!--l. 25--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-80001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | log2(35775.6) = <span
| + | |
| − | class="cmbx-12">15.1 cycles </span>(assuming perfect PCR)
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-90002"></a>Modified PCR using post-elution primers</h3>
| + | |
| − | <!--l. 28--><p class="noindent" >Redo PCR using same amounts of reagents, but with 20 cycles instead of 30.
| + | |
| − | Made 2x 50 uL for each MaSp plasmid.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/30/2015 - Post-elution PCR amplification of MaSp plasmids</summary>
| + | |
| − | <head><title>4/30/2015</title>
| + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html --> | + | |
| − | <meta name="src" content="4_30_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-18 23:23:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_30_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/30/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Post-elution primer PCR of MaSp plasmids</h3>
| + | |
| − | <!--l. 14--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | The previous anomalous results may be due to a human factor. So we | + | |
| − | decided to have Olivia prepare the PCR today using the same protocol,
| + | |
| − | but scaled to 3x 25 uL each for 2AB and 2BC. Results shown below:
| + | |
| − | <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a | + | |
| − | id="x1-2001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 18--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/7/71/4_30_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Post-elution primer PCR of MaSp plasmids. This result is very
| + | |
| − | strange.</span></div><!--tex4ht:label?: x1-2001r1 -->
| + | |
| − | | + | |
| − | <!--l. 20--><p class="indent" > </div><hr class="endfigure">
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/29/2015 - Post-elution PCR amplification of MaSp plasmids</summary>
| + | |
| − | <head><title>4/29/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="4_29_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-18 23:18:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_29_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/29/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Post-Elution primer PCR of MaSp 2 plasmids</h3>
| + | |
| − | <!--l. 13--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Performed same PCR reaction as yesterday, but with 2x 50 uL for | + | |
| − | each reaction. This was prepared as a master mix. Results shown below.
| + | |
| − | <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a | + | |
| − | id="x1-20011"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 17--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/e/e7/4_29_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Post-elution amplification of MaSp plasmids. Results are
| + | |
| − | inconsistent even though a master mix was used.</span></div><!--tex4ht:label?: x1-20011 -->
| + | |
| − | | + | |
| − | <!--l. 19--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 20--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | We excised the bands from 2AB, 2BC (only one), and 2CA.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/28/2015 - Post-elution PCR amplification of MaSp plasmids</summary>
| + | |
| − | <head><title>4/28/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="4_28_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-18 23:11:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_28_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/28/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Post-Elution Primer PCR Amplification of MaSp2 Sequences</h3>
| + | |
| − | <!--l. 14--><p class="noindent" >We used the post-elution primers to test amplify 2AB plasmid and to determine
| + | |
| − | an appropriate annealing temperature. We used the following protocol:
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 15--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">1x Reaction (uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">4x Reaction (uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">5x Q5 Buffer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">20 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">10 mM dNTPs </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">0.5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">2 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">10 uM For (F-03) </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">1.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-3"
| + | |
| − | class="td11">5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">10 uM Rev (G-03)</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">1.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-3"
| + | |
| − | class="td11">5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11">2AB (500 pg) </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11">1.74 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-3"
| + | |
| − | class="td11">6.96 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11">5x GC buffer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11">5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-3"
| + | |
| − | class="td11">20 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-1"
| + | |
| − | class="td11">Q5 Pol </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-2"
| + | |
| − | class="td11">0.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-3"
| + | |
| − | class="td11">1 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-2"
| + | |
| − | class="td11">10.01 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-3"
| + | |
| − | class="td11">40.04 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-10-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">25 </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">100 </span></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" /> | + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 29--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11">30 sec</td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">10 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-1"
| + | |
| − | class="td11">69, 66.8, 65.4, 63 C</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-2"
| + | |
| − | class="td11">15 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-2"
| + | |
| − | class="td11">15 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-1"
| + | |
| − | class="td11">Repeat from step 2</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-2"
| + | |
| − | class="td11">30 x </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-2"
| + | |
| − | class="td11">2 min</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-1"
| + | |
| − | class="td11">12 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-2"
| + | |
| − | class="td11">hold </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a | + | |
| − | id="x1-20002"></a>Results</h3>
| + | |
| − | <!--l. 41--><p class="noindent" ><hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-2001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 43--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/0/07/4_28_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Annealing temperature test with MaSp 2AB at different
| + | |
| − | temperatures. Our expected product is 170 bp.</span></div><!--tex4ht:label?: x1-2001r1 -->
| + | |
| − | | + | |
| − | <!--l. 45--><p class="noindent" ></div><hr class="endfigure">
| + | |
| − | <!--l. 46--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-30002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Base on these results, we decided to scale up to 3x 50 uL reactions for each
| + | |
| − | monomer, and would use 66 C as the annealing temperature.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">3 </span> <a
| + | |
| − | id="x1-40003"></a>Amplification Again</h3>
| + | |
| − | <!--l. 49--><p class="noindent" >We scaled up the above protocol for 3x 50 uL reactions for each monomer.
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 50--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-4" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-4-1g"><col
| + | |
| − | id="TBL-4-1"><col
| + | |
| − | id="TBL-4-2"><col
| + | |
| − | id="TBL-4-3"><col
| + | |
| − | id="TBL-4-4"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:center;" id="TBL-4-1-2"
| + | |
| − | class="td11">AB (287 pg/uL)</td><td style="white-space:nowrap; text-align:center;" id="TBL-4-1-3"
| + | |
| − | class="td11">BC (260 pg/uL)</td><td style="white-space:nowrap; text-align:center;" id="TBL-4-1-4"
| + | |
| − | class="td11">CA (223 pg/uL)</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-1"
| + | |
| − | class="td11">5x Q5 Buffer </td><td colspan="3" style="white-space:nowrap; text-align:center;" id="TBL-4-2-2"
| + | |
| − | class="td11"> <div class="multicolumn" style="white-space:nowrap; text-align:center;">30 uL</div>
| + | |
| − | </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-1"
| + | |
| − | class="td11">10 mM dNTPs </td><td colspan="3" style="white-space:nowrap; text-align:center;" id="TBL-4-3-2"
| + | |
| − | class="td11"> <div class="multicolumn" style="white-space:nowrap; text-align:center;">3 uL</div>
| + | |
| − | </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-4-1"
| + | |
| − | class="td11">10 uM For (F-03) </td><td colspan="3" style="white-space:nowrap; text-align:center;" id="TBL-4-4-2"
| + | |
| − | class="td11"> <div class="multicolumn" style="white-space:nowrap; text-align:center;">7.5 uL</div>
| + | |
| − | </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-5-1"
| + | |
| − | class="td11">10 uM Rev (G-03)</td><td colspan="3" style="white-space:nowrap; text-align:center;" id="TBL-4-5-2"
| + | |
| − | class="td11"> <div class="multicolumn" style="white-space:nowrap; text-align:center;">7.5 uL</div>
| + | |
| − | </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-6-1"
| + | |
| − | class="td11">Template (500 pg)</td><td style="white-space:nowrap; text-align:center;" id="TBL-4-6-2"
| + | |
| − | class="td11"> 5.22 uL </td><td style="white-space:nowrap; text-align:center;" id="TBL-4-6-3"
| + | |
| − | class="td11"> 5.76 uL </td><td style="white-space:nowrap; text-align:center;" id="TBL-4-6-4"
| + | |
| − | class="td11"> 6.73 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-7-1"
| + | |
| − | class="td11">Q5 Polymerase </td><td colspan="3" style="white-space:nowrap; text-align:center;" id="TBL-4-7-2"
| + | |
| − | class="td11"> <div class="multicolumn" style="white-space:nowrap; text-align:center;">1.5 uL</div>
| + | |
| − | </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-8-1"
| + | |
| − | class="td11">5x GC enhancer </td><td colspan="3" style="white-space:nowrap; text-align:center;" id="TBL-4-8-2"
| + | |
| − | class="td11"> <div class="multicolumn" style="white-space:nowrap; text-align:center;">30 uL</div>
| + | |
| − | </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-9-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:center;" id="TBL-4-9-2"
| + | |
| − | class="td11"> 65.28 uL </td><td style="white-space:nowrap; text-align:center;" id="TBL-4-9-3"
| + | |
| − | class="td11"> 64.74 uL </td><td style="white-space:nowrap; text-align:center;" id="TBL-4-9-4"
| + | |
| − | class="td11"> 63.77 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-10-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-10-1"
| + | |
| − | class="td11">Total </td><td colspan="3" style="white-space:nowrap; text-align:center;" id="TBL-4-10-2"
| + | |
| − | class="td11"> <div class="multicolumn" style="white-space:nowrap; text-align:center;">150 uL</div></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 64--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-5" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-5-1g"><col
| + | |
| − | id="TBL-5-1"><col
| + | |
| − | id="TBL-5-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-1-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-1-2"
| + | |
| − | class="td11">30 sec</td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-2-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-2-2"
| + | |
| − | class="td11">10 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-3-1"
| + | |
| − | class="td11">66 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-3-2"
| + | |
| − | class="td11">15 sec</td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-4-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-4-2"
| + | |
| − | class="td11">15 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-5-1"
| + | |
| − | class="td11">repeat from step 2</td><td style="white-space:nowrap; text-align:left;" id="TBL-5-5-2"
| + | |
| − | class="td11">30x </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-6-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-6-2"
| + | |
| − | class="td11">2 min</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-7-1"
| + | |
| − | class="td11">12 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-7-2"
| + | |
| − | class="td11">hold </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">4 </span> <a
| + | |
| − | id="x1-50004"></a>Results for second PCR</h3>
| + | |
| − | <!--l. 76--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-60004"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Visualized on 2% gel. Used 2 uL 50 bp ladder. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-6001r2"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 80--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/b/b6/4_28_2015_2.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 2: </span><span
| + | |
| − | class="content">Results from large scale amplification of MaSp using post-elution
| + | |
| − | primers. The amplification is inconsistent.</span></div><!--tex4ht:label?: x1-6001r2 -->
| + | |
| − | | + | |
| − | <!--l. 82--><p class="indent" > </div><hr class="endfigure">
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/27/2015 - Initial ICA Ligation Test</summary>
| + | |
| − | <head><title>4/27/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="4_27_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-18 23:07:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_27_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/27/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>2AB, 2BC, 2CA Sticky End Ligation Test</h3>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 14--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"><col
| + | |
| − | id="TBL-2-4"><col
| + | |
| − | id="TBL-2-5"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">AB+BC</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">BC+CA</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">CA+AB</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-5"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">AB+BC+CA</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">2x T7 Ligase Buffer</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-4"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-5"
| + | |
| − | class="td11">5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">1.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">1.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-4"
| + | |
| − | class="td11">0.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-5"
| + | |
| − | class="td11">0 uL </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">DNA 1 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-3"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-4"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-5"
| + | |
| − | class="td11">2 uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">DNA 2 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-3"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-4"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-5"
| + | |
| − | class="td11">1 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11">DNA 3 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11">n/a </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-3"
| + | |
| − | class="td11">n/a </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-4"
| + | |
| − | class="td11">n/a </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-5"
| + | |
| − | class="td11">2 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11">T7 Ligase </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11">0.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-3"
| + | |
| − | class="td11">0.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-4"
| + | |
| − | class="td11">0.5 ul </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-5"
| + | |
| − | class="td11">0.5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">10 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">10 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">10 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-5"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">10 uL </span></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 26--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11">25 C</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11">5 min</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">65 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11"> </td></tr></table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 32--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | The ligation test was run on 2% gel with 2 uL of 50 bp ladder. Results shown
| + | |
| − | below. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-2001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 36--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/thumb/f/fb/4_27_2015.jpg/800px-4_27_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">T7 Ligation Test with MaSp2 fragments. BC was used as a
| + | |
| − | negative ligation control. The presence of ladders may indicate non-specific
| + | |
| − | binding.</span></div><!--tex4ht:label?: x1-2001r1 -->
| + | |
| − | | + | |
| − | <!--l. 38--><p class="indent" > </div><hr class="endfigure">
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| | </details> | | </details> |
| − |
| |
| | <details> | | <details> |
| | <summary>4/19/2015 - 4/25/2015</summary> | | <summary>4/19/2015 - 4/25/2015</summary> |
| − | <details> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/24_April_2015"><li>4/24/2015 - Sending DNA for sequencing</li></a> |
| − | <summary>4/24/2015 - Sending DNA for sequencing</summary>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/23_April_2015"><li>4/23/2015 - Miniprep DNA for sequencing</li></a> |
| − | <head><title>4/24/2015</title>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/22_April_2015"><li>4/22/2015 - Colony PCR</li></a> |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/21_April_2015"><li>4/21/2015 - Ligation of MaSp sequences into pSB1C3 and Transformation</li></a> |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/20_April_2015"><li>4/20/2015 - PCR amplification of pSB1C3 and EcoRI, PstI digestion</li></a> |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
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| − | <meta name="src" content="4_24_2015.tex">
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| − | <meta name="date" content="2015-05-18 23:00:00">
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| − | <link rel="stylesheet" type="text/css" href="4_24_2015.css">
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| − | </head><body
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| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/24/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Sequencing of MaSp2 constructs</h3>
| + | |
| − | <!--l. 13--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Prepared each sample from yesterday for sequencing.
| + | |
| − | <!--l. 15--><p class="noindent" ><span class="paragraphHead"><a | + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Diluted 500 ng of each DNA species and 2.5 uL of VF2 primer into 15 uL in a
| + | |
| − | PCR strip.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a | + | |
| − | id="x1-40002"></a>Results: 4/25/2015</h3>
| + | |
| − | <!--l. 18--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-50002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Used Clustal W2 to align sequencing results.
| + | |
| − | | + | |
| − | <!--l. 20--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-60002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Constructs 2AB-2, 2BC-1, and 2CA-2 are sequenced properly.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/23/2015 - Miniprep DNA for sequencing</summary>
| + | |
| − | <head><title>4/23/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
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| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
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| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
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| − | <meta name="src" content="4_23_2015.tex">
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| − | <meta name="date" content="2015-05-18 22:56:00">
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| − | </head><body
| + | |
| − | >
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| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/23/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Miniprep</h3>
| + | |
| − | <!--l. 13--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Prepared Glycerol stocks of each culture: 500 uL of bacteria + 500 uL 50% | + | |
| − | v/v glycerol.
| + | |
| − | <!--l. 15--><p class="noindent" ><span class="paragraphHead"><a | + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Used zymo miniprep kit to extract DNA from yesterday’s cells. <div class="table">
| + | |
| − | | + | |
| − | <!--l. 17--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Concentration (ng/uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">2AB-1</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">640 </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">2AB-2</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">287</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">2BC-1</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">261 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">2BC-2</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">275 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11">2CA-1</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11">469 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11">2CA-2</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11">223 </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/22/2015 - Colony PCR</summary>
| + | |
| − | <head><title>4/22/2015</title>
| + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
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| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
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| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
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| − | <!-- html --> | + | |
| − | <meta name="src" content="4_22_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-18 22:48:00">
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| − | <link rel="stylesheet" type="text/css" href="4_22_2015.css">
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| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/22/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Bacterial Transformation</h3>
| + | |
| − | <!--l. 14--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Observed many colonies growing on the plates. We will do colony PCR to
| + | |
| − | verify proper insertion.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-30002"></a>Colony PCR</h3>
| + | |
| − | <!--l. 17--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-40002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Picked four colonies from each plate. Resuspended each each colony in 100 uL | + | |
| − | water. Used protocol below: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 19--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">1x Reaction (uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">12x Reaction (uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">5x Q5 buffer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">60 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">10 mM dNTPs </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">0.5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">6 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">10 uM For (VF2) </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">1.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-3"
| + | |
| − | class="td11">15 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">10 uM Rev (post-elution G-03)</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">1.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-3"
| + | |
| − | class="td11">15 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11">Colony (Resuspended) </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-3"
| + | |
| − | class="td11">1 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11">5x GC Enhancer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11">5 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-3"
| + | |
| − | class="td11">60 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-1"
| + | |
| − | class="td11">Q5 polymerase </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-2"
| + | |
| − | class="td11">0.25 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-3"
| + | |
| − | class="td11">3 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-2"
| + | |
| − | class="td11">10.75 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-3"
| + | |
| − | class="td11">129 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-10-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">25 </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">300 </span></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 33--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11">3 min</td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">10 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-1"
| + | |
| − | class="td11">66 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-2"
| + | |
| − | class="td11">15 sec</td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-2"
| + | |
| − | class="td11">15 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-1"
| + | |
| − | class="td11">repeat from step 2</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-2"
| + | |
| − | class="td11">25 x </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-2"
| + | |
| − | class="td11">2 min</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-1"
| + | |
| − | class="td11">12 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-2"
| + | |
| − | class="td11">hold </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">3 </span> <a | + | |
| − | id="x1-50003"></a>Results</h3>
| + | |
| − | <!--l. 45--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-60003"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Cast 1.0% gel to visualize. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-6001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 49--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/thumb/3/3f/4_22_2015.jpg/800px-4_22_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Colony PCR to verify incorporation of insert.</span></div><!--tex4ht:label?: x1-6001r1 -->
| + | |
| − | | + | |
| − | <!--l. 51--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 52--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-70003"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | We picked colonies 3 and 4 from each construct and grew 5 mL liquid culture
| + | |
| − | for miniprep and sequencing tomorrow. Renumbered the cultures to 1 and 2
| + | |
| − | respectively.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">4 </span> <a
| + | |
| − | id="x1-80004"></a>Column purification of MaSp2 PCA</h3>
| + | |
| − | <!--l. 55--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-90004"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Used zymo kit to purify yesterday’s MaSp2 PCA reactions. Eluted in 10 uL
| + | |
| − | EB. Results below: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 57--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-4" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-4-1g"><col
| + | |
| − | id="TBL-4-1"><col
| + | |
| − | id="TBL-4-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Concentration (ng/uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-1"
| + | |
| − | class="td11">2AB</td><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-2"
| + | |
| − | class="td11">279.34 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-1"
| + | |
| − | class="td11">2BC</td><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-2"
| + | |
| − | class="td11">154.57 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-4-1"
| + | |
| − | class="td11">2CA</td><td style="white-space:nowrap; text-align:left;" id="TBL-4-4-2"
| + | |
| − | class="td11">236.36 </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">5 </span> <a
| + | |
| − | id="x1-100005"></a>BsaI Digestion of PCA products from 4/21/2015</h3>
| + | |
| − | <!--l. 66--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-110005"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Set up 2x 25 uL reactions for each: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 68--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-5" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-5-1g"><col
| + | |
| − | id="TBL-5-1"><col
| + | |
| − | id="TBL-5-2"><col
| + | |
| − | id="TBL-5-3"><col
| + | |
| − | id="TBL-5-4"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2AB(279 ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-5-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2BC (155 ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-5-1-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2CA (237 ng/uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-2-1"
| + | |
| − | class="td11">DNA </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-2-2"
| + | |
| − | class="td11">2 uL ( 560 ng) </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-2-3"
| + | |
| − | class="td11">5 uL ( 770 ng) </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-2-4"
| + | |
| − | class="td11">2.5 uL ( 590 ng) </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-3-1"
| + | |
| − | class="td11">10x Cutsmart</td><td style="white-space:nowrap; text-align:left;" id="TBL-5-3-2"
| + | |
| − | class="td11">2.5 uL: </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-3-3"
| + | |
| − | class="td11">2.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-3-4"
| + | |
| − | class="td11">2.5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-4-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-4-2"
| + | |
| − | class="td11">18.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-4-3"
| + | |
| − | class="td11">15.5 uL: </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-4-4"
| + | |
| − | class="td11">18 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-5-1"
| + | |
| − | class="td11">BsaI </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-5-2"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-5-3"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-5-4"
| + | |
| − | class="td11">2 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-6-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-5-6-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">25 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-5-6-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">25 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-5-6-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">25 uL </span></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 78--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-6" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-6-1g"><col
| + | |
| − | id="TBL-6-1"><col
| + | |
| − | id="TBL-6-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-6-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-6-1-1"
| + | |
| − | class="td11">50 C</td><td style="white-space:nowrap; text-align:left;" id="TBL-6-1-2"
| + | |
| − | class="td11">2 hr </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-6-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-6-2-1"
| + | |
| − | class="td11">65 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-6-2-2"
| + | |
| − | class="td11">20 min</td></tr></table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 84--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-120005"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | The digestion products were column purified using zymo kit with the following
| + | |
| − | yields: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 86--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-7" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-7-1g"><col
| + | |
| − | id="TBL-7-1"><col
| + | |
| − | id="TBL-7-2"><col
| + | |
| − | id="TBL-7-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-7-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-7-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-7-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Concentration (ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-7-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">A 260/280</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-7-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-7-2-1"
| + | |
| − | class="td11">2AB</td><td style="white-space:nowrap; text-align:left;" id="TBL-7-2-2"
| + | |
| − | class="td11">24.7 </td><td style="white-space:nowrap; text-align:left;" id="TBL-7-2-3"
| + | |
| − | class="td11">2.09 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-7-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-7-3-1"
| + | |
| − | class="td11">2BC</td><td style="white-space:nowrap; text-align:left;" id="TBL-7-3-2"
| + | |
| − | class="td11">55.17 </td><td style="white-space:nowrap; text-align:left;" id="TBL-7-3-3"
| + | |
| − | class="td11">1.9 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-7-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-7-4-1"
| + | |
| − | class="td11">2CA</td><td style="white-space:nowrap; text-align:left;" id="TBL-7-4-2"
| + | |
| − | class="td11">22.38 </td><td style="white-space:nowrap; text-align:left;" id="TBL-7-4-3"
| + | |
| − | class="td11">2.14 </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/21/2015 - Ligation of MaSp sequences into pSB1C3 and Transformation</summary>
| + | |
| − | <head><title>4/21/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="4_21_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-18 22:44:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_21_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/21/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Ligation of MaSp 2AB, 2BC, 2CA in to pSB1C3</h3>
| + | |
| − | <!--l. 13--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Used ligation calculator to determine amounts of each insert to use to ligate | + | |
| − | into 50 ng of vector for a 3:1 ratio of insert to vector.
| + | |
| − | <!--l. 15--><p class="noindent" ><span class="paragraphHead"><a | + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | 2000 bp vector, 170 bp insert.
| + | |
| − | <!--l. 17--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-40001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Determined 12.75 ng of insert to 50 ng vector. Proceeded with ligation using
| + | |
| − | protocol below: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 19--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"><col
| + | |
| − | id="TBL-2-4"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2AB (43.28 ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2BC (34.56 ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2CA (34.9 ng/uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">T4 Ligase </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-4"
| + | |
| − | class="td11">1 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">Insert (12.75 ng) </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">0.30 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">0.370 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-4"
| + | |
| − | class="td11">0.365 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">Vector (50 ng) </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">1.484 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-3"
| + | |
| − | class="td11">1.484 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-4"
| + | |
| − | class="td11">1.484 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">10x T4 Ligase Buffer</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-3"
| + | |
| − | class="td11">2 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-4"
| + | |
| − | class="td11">2 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11">15.22 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-3"
| + | |
| − | class="td11">15.15 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-4"
| + | |
| − | class="td11">15.16 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">20 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">20 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">20 uL </span></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 30--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11">25 C</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11">20 min</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">65 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">10 min</td></tr></table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-50002"></a>Bacterial Transformation</h3>
| + | |
| − | <!--l. 37--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-60002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Used 2 uL of each reaction to transform 50 uL competent cells.
| + | |
| − | <!--l. 39--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-70002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Incubate on ice 5 min.
| + | |
| − | <!--l. 41--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-80002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Add 200 uL of pre-warmed SOC to each tube.
| + | |
| − | <!--l. 43--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-90002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Rescue for 10 min.
| + | |
| − | <!--l. 45--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-100002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Plate 50 uL onto chloramphenicol plates. Incubate at 37 C overnight.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">3 </span> <a
| + | |
| − | id="x1-110003"></a>MaSp2 PCA</h3>
| + | |
| − | <!--l. 48--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-120003"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Set up 2x 50 uL reactions for AB, BC, CA each using protocol from 4/13/2015
| + | |
| − | scaled to 50 uL with the following modifications:
| + | |
| − | <!--l. 50--><p class="indent" > <span class="subparagraphHead"> <a
| + | |
| − | id="x1-130003"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span> Annealing temp: 64 C
| + | |
| − | <!--l. 52--><p class="indent" > <span class="subparagraphHead"> <a
| + | |
| − | id="x1-140003"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span> 25 cycles instead of 20.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/20/2015 - PCR amplification of pSB1C3 and EcoRI, PstI digestion</summary>
| + | |
| − | <head><title>4/20/2015</title>
| + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="4_20_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-18 22:39:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_20_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/20/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>PCR Amplification of pSB1C3</h3>
| + | |
| − | <!--l. 14--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Due to poor amplification of pSB1C3 from friday, we are doing a temperature
| + | |
| − | test today.
| + | |
| − | <!--l. 16--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Followed same protocol as 4/17/2015, but scaled to 3x 25 uL reactions.
| + | |
| − | <!--l. 18--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-40001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Decided to take nanodrop reading of the template Julian provided. It was 230
| + | |
| − | ng/uL. Instead of using stock concentration of template, we will use a 1:100
| + | |
| − | dilution. Results shown below. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-4001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 22--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/9/90/4_20_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">pSB1C3 amplification temperature test</span></div><!--tex4ht:label?: x1-4001r1 -->
| + | |
| − | | + | |
| − | <!--l. 24--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 25--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-50001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | We gel extracted the bands together. 26.4 ng/uL, A 260/280: 1.56.
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-60002"></a>EcoRI and PstI Digestion of MaSp 2AB, 2BC, 2CA, and pSB1C3</h3>
| + | |
| − | <!--l. 28--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-70002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | We pooled together all the linearized pSB1C3 that we had. Final concentration:
| + | |
| − | 16.5 ng/uL.
| + | |
| − | <!--l. 30--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-80002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Set up digestion as below: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 32--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Concentration (ng/uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">2AB </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">37.58 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">2BC </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">42.8 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">2CA </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">79.11 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">pSB1C3</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">16.5 </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 41--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"><col
| + | |
| − | id="TBL-3-3"><col
| + | |
| − | id="TBL-3-4"><col
| + | |
| − | id="TBL-3-5"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2AB </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2BC </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">2CA </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-5"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">pSB1C3</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">EcoRI </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-3"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-4"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-5"
| + | |
| − | class="td11">1 uL </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-1"
| + | |
| − | class="td11">PstI </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-2"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-3"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-4"
| + | |
| − | class="td11">1 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-5"
| + | |
| − | class="td11">1uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-1"
| + | |
| − | class="td11">10x NEbuffer 2.1</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-2"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-3"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-4"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-5"
| + | |
| − | class="td11">5 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-1"
| + | |
| − | class="td11">DNA (500 ng) </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-2"
| + | |
| − | class="td11">16 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-3"
| + | |
| − | class="td11">11 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-4"
| + | |
| − | class="td11">6 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-5"
| + | |
| − | class="td11">19 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-2"
| + | |
| − | class="td11">27 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-3"
| + | |
| − | class="td11">32 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-4"
| + | |
| − | class="td11">37 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-5"
| + | |
| − | class="td11">24 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-4"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-5"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 uL </span></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 52--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-4" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-4-1g"><col
| + | |
| − | id="TBL-4-1"><col
| + | |
| − | id="TBL-4-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-1"
| + | |
| − | class="td11">37 C</td><td style="white-space:nowrap; text-align:left;" id="TBL-4-1-2"
| + | |
| − | class="td11">1 hr </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-1"
| + | |
| − | class="td11">65 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-2-2"
| + | |
| − | class="td11">20 min</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-4-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-1"
| + | |
| − | class="td11">12 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-4-3-2"
| + | |
| − | class="td11">hold </td></tr></table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <!--l. 59--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-90002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Column purified all DNA after digestion using Zymo kit. Eluted in 10 uL of
| + | |
| − | EB. Results shown below: <div class="table">
| + | |
| − | | + | |
| − | <!--l. 61--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-5" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-5-1g"><col
| + | |
| − | id="TBL-5-1"><col
| + | |
| − | id="TBL-5-2"><col
| + | |
| − | id="TBL-5-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Concentration (ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-5-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">A 260/280</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-2-1"
| + | |
| − | class="td11">2AB </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-2-2"
| + | |
| − | class="td11">43.28 </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-2-3"
| + | |
| − | class="td11">1.9 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-3-1"
| + | |
| − | class="td11">2BC </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-3-2"
| + | |
| − | class="td11">34.56 </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-3-3"
| + | |
| − | class="td11">1.7 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-4-1"
| + | |
| − | class="td11">2CA </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-4-2"
| + | |
| − | class="td11">34.9 </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-4-3"
| + | |
| − | class="td11">1.7 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-5-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-5-5-1"
| + | |
| − | class="td11">pSB1C3</td><td style="white-space:nowrap; text-align:left;" id="TBL-5-5-2"
| + | |
| − | class="td11">33.7 </td><td style="white-space:nowrap; text-align:left;" id="TBL-5-5-3"
| + | |
| − | class="td11">1.61 </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| | </details> | | </details> |
| − |
| |
| | <details> | | <details> |
| | <summary>4/12/2015 - 4/18/2015</summary> | | <summary>4/12/2015 - 4/18/2015</summary> |
| − | <details> | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/17_April_2015"><li>4/17/2015 - Amplification of pSB1C3</li></a> |
| − | <summary>4/17/2015 - Amplification of pSB1C3</summary>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/16_April_2015"><li>4/16/2015 - PCA of MaSp2 Pieces</li></a> |
| − | <head><title>4/17/2015</title>
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/15_April_2015"><li>4/15/2015 - PCA of MaSp2 Pieces</li></a> |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/14_April_2015"><li>4/14/2015 - PCA of MaSp2 Pieces</li></a> |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/13_April_2015"><li>4/13/2015 - PCA of MaSp2 Pieces</li></a> |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html --> | + | |
| − | <meta name="src" content="4_17_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-18 22:32:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_17_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/17/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>PCR amplification of pSB1C3</h3>
| + | |
| − | <!--l. 14--><p class="noindent" >We set up a 50 uL reaction using the following protocol from Julian’s notebook.
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 15--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Volume (uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">5x Q5 Buffer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">10 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">10 mM dNTPs </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">1 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">10 uM For (SB-prep 2EA) </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">2.5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">10 uM Rev (SB-prep 3P1) </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">2.5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11">Template (Spycatcher from Julian)</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11">1 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11">Q5 Polymerase </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11">.0.5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-2"
| + | |
| − | class="td11">32.5 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">50 </span></td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 28--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-3" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-3-1g"><col
| + | |
| − | id="TBL-3-1"><col
| + | |
| − | id="TBL-3-2"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-1-2"
| + | |
| − | class="td11">30 sec</td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-2-2"
| + | |
| − | class="td11">10 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-1"
| + | |
| − | class="td11">66 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-3-2"
| + | |
| − | class="td11">20 sec</td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-4-2"
| + | |
| − | class="td11">30 sec</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-1"
| + | |
| − | class="td11">repeat from step 2</td><td style="white-space:nowrap; text-align:left;" id="TBL-3-5-2"
| + | |
| − | class="td11">30 x </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-6-2"
| + | |
| − | class="td11">2 min</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-3-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-1"
| + | |
| − | class="td11">12 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-3-7-2"
| + | |
| − | class="td11">hold </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-20002"></a>Results</h3>
| + | |
| − | <!--l. 40--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-30002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Cast a 1% gel to visualize. <hr class="figure"><div class="figure" | + | |
| − | >
| + | |
| − | | + | |
| − | <a | + | |
| − | id="x1-3001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 44--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/1/15/4_17_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">PCR amplification of pSB1C3</span></div><!--tex4ht:label?: x1-3001r1 -->
| + | |
| − | | + | |
| − | <!--l. 46--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 47--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-40002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Gel extraction (Qiagen) yielded 36.47 ng/uL, A 260/280: 1.91.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/16/2015 - PCA of MaSp2 Pieces</summary>
| + | |
| − | <head><title>4/16/2015</title> | + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="4_16_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-17 23:04:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_16_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/16/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Polymerase Chain Assembly of MaSp 2AB sequence.</h3>
| + | |
| − | <!--l. 14--><p class="noindent" >Repeated PCA of 2AB using protocol from 4/13/2015 with the follow
| + | |
| − | modifications.
| + | |
| − | <!--l. 15--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Annealing Temp: 64 C | + | |
| − | <!--l. 17--><p class="noindent" ><span class="paragraphHead"><a | + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | 25 cycles instead of 20.
| + | |
| − | <!--l. 19--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-40001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | We cast a 1.5% gel to visualize the results shown below. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-4001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 23--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/d/d7/4_16_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">PCA of MaSp 2AB. Our target product is at 170 bp.</span></div><!--tex4ht:label?: x1-4001r1 -->
| + | |
| − | | + | |
| − | <!--l. 25--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-50002"></a>Gel Extraction</h3>
| + | |
| − | <!--l. 27--><p class="noindent" >Fasih gel extracted the two 2AB bands. <div class="table">
| + | |
| − | | + | |
| − | <!--l. 28--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Amount (ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">A 260/280</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">2AB-1</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">46.28 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">2.41 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">2AB-2</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">24.97 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">4.29 </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/15/2015 - PCA of MaSp2 Pieces</summary>
| + | |
| − | <head><title>4/15/2015</title>
| + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="4_15_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-17 21:07:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_15_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/15/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>MaSp2 Polymerase Chain Assembly</h3>
| + | |
| − | <!--l. 13--><p class="noindent" >Set up two 50 uL PCR reactions each for 2BC and 2CA using the same guidelines
| + | |
| − | on 4/13/2015 with the following modifications:
| + | |
| − | <!--l. 14--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Annealing Temp: 64 C | + | |
| − | <!--l. 16--><p class="noindent" ><span class="paragraphHead"><a | + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | 25 cycles instead of 20
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-40002"></a>Gel Purification of MaSp2 Monomers</h3>
| + | |
| − | <!--l. 19--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-50002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Visualized with 1.5% agarose gel. Similar result to yesterday’s gel.
| + | |
| − | | + | |
| − | <!--l. 21--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-60002"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Used gel purification kit (zymo) to extract DNA from 2AB, 2BC, and 2CA.
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 23--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Amount (ng/uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">A 260/280</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">2AB</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">12.73 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">2.01 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">2BC</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">42.8 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">1.93 </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">2CA</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">79.11 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-3"
| + | |
| − | class="td11">1.91 </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/14/2015 - PCA of MaSp2 Pieces</summary>
| + | |
| − | <head><title>4/14/2015</title>
| + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="4_14_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-15 21:17:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_14_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/14/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>MaSp2 Polymerase Chain Assembly</h3>
| + | |
| − | <!--l. 14--><p class="noindent" >Set up 2 50 uL reactions following the same protocol from yesterday scaled to 50
| + | |
| − | uL for 2AB. We included the following modifications:
| + | |
| − | <!--l. 15--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Annealing temperature: 64 C | + | |
| − | <!--l. 17--><p class="noindent" ><span class="paragraphHead"><a | + | |
| − | id="x1-30001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | 25 cycles instead of 20
| + | |
| − | <!--l. 19--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-40001"></a><span
| + | |
| − | class="cmbsy-10x-x-120">∙</span></span>
| + | |
| − | Used primers 2A-F, 2B-R
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-50002"></a>Results</h3>
| + | |
| − | <!--l. 22--><p class="noindent" >Cast 1.5% TAE agarose gel. Used 3 uL of NEB 50 bp ladder. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-5001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 25--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/1/1f/4_14_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Each band corresponds to 25 uL reaction of MaSp2. </span></div><!--tex4ht:label?: x1-5001r1 -->
| + | |
| − | | + | |
| − | <!--l. 27--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 28--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-60002"></a></span>
| + | |
| − | This protocol works better than the one previously. The target product at 170
| + | |
| − | bp is clearly more intense. While there is still some primer dimer formation, we
| + | |
| − | will stick with this protocol.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>4/13/2015 - PCA of MaSp2 Pieces</summary>
| + | |
| − | <head><title>4/13/2015</title>
| + | |
| − | <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
| + | |
| − | <meta name="generator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <meta name="originator" content="TeX4ht (http://www.cse.ohio-state.edu/~gurari/TeX4ht/)">
| + | |
| − | <!-- html -->
| + | |
| − | <meta name="src" content="4_13_2015.tex">
| + | |
| − | <meta name="date" content="2015-05-14 12:35:00">
| + | |
| − | <link rel="stylesheet" type="text/css" href="4_13_2015.css">
| + | |
| − | </head><body
| + | |
| − | >
| + | |
| − | <div class="maketitle">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h2 class="titleHead">4/13/2015</h2>
| + | |
| − | <div class="author" ></div><br />
| + | |
| − | <div class="date" ></div>
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">1 </span> <a
| + | |
| − | id="x1-10001"></a>Polymerase Chain Assembly</h3>
| + | |
| − | <!--l. 14--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-20001"></a></span>
| + | |
| − | The primers corresponding to the new MaSp2 sequences with a valine added
| + | |
| − | to the end came today. We conducted polymerase chain assembly to place the
| + | |
| − | biobrick prefix/suffix and the BsaI restriction enzyme recognition sites with the
| + | |
| − | designed sticky ends.
| + | |
| − | <!--l. 16--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-30001"></a></span>
| + | |
| − | We used NEB’s Q5 PCR annealing temperature calculator to determine that
| + | |
| − | an annealing temperature of 65 C was optimal. We also decided to test
| + | |
| − | annealing at 61.4 C and 59 C. In addition, we planned to use the GC
| + | |
| − | enhancer because our sequence is GC-rich. We prepared reactions as follows:
| + | |
| − | <div class="table">
| + | |
| − | | + | |
| − | <!--l. 18--><p class="indent" > <hr class="float"><div class="float"
| + | |
| − | >
| + | |
| − | | + | |
| − | <div class="tabular"> <table id="TBL-2" class="tabular"
| + | |
| − | cellspacing="0" cellpadding="0"
| + | |
| − | ><colgroup id="TBL-2-1g"><col
| + | |
| − | id="TBL-2-1"><col
| + | |
| − | id="TBL-2-2"><col
| + | |
| − | id="TBL-2-3"></colgroup><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-1-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-1"
| + | |
| − | class="td11"> </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">1x (uL)</span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-1-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">3x (uL)</span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-2-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-1"
| + | |
| − | class="td11">5x Q5 Buffer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-2"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-2-3"
| + | |
| − | class="td11">15 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-3-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-1"
| + | |
| − | class="td11">10 mM dNTPs </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-2"
| + | |
| − | class="td11">0.5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-3-3"
| + | |
| − | class="td11">1.5 uL </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-4-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-1"
| + | |
| − | class="td11">10 uM For </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-2"
| + | |
| − | class="td11">1.25 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-4-3"
| + | |
| − | class="td11">3.75 uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-5-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-1"
| + | |
| − | class="td11">10 uM Rev </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-2"
| + | |
| − | class="td11">1.25 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-5-3"
| + | |
| − | class="td11">3.75 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-6-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-1"
| + | |
| − | class="td11">MaSp core (2.7 pg/uL)</td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-2"
| + | |
| − | class="td11">3.7 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-6-3"
| + | |
| − | class="td11">11.1 uL </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-7-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-1"
| + | |
| − | class="td11">5x GC enhancer </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-2"
| + | |
| − | class="td11">5 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-7-3"
| + | |
| − | class="td11">15 uL </td></tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-8-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-1"
| + | |
| − | class="td11">Q5 Polymerase </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-2"
| + | |
| − | class="td11">0.25 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-8-3"
| + | |
| − | class="td11">0.75 uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-9-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-1"
| + | |
| − | class="td11">ddH2O </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-2"
| + | |
| − | class="td11">8.05 uL </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-9-3"
| + | |
| − | class="td11">24.15 uL</td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-10-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-1"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">Total </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-2"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">25 uL </span></td><td style="white-space:nowrap; text-align:left;" id="TBL-2-10-3"
| + | |
| − | class="td11"><span
| + | |
| − | class="cmbx-12">75 uL </span></td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-11-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-11-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-11-2"
| + | |
| − | class="td11">30 s </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-11-3"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-12-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-12-1"
| + | |
| − | class="td11">98 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-12-2"
| + | |
| − | class="td11">10 s </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-12-3"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-13-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-13-1"
| + | |
| − | class="td11">65/61.4/59 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-13-2"
| + | |
| − | class="td11">15 s </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-13-3"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-14-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-14-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-14-2"
| + | |
| − | class="td11">15 s </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-14-3"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-15-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-15-1"
| + | |
| − | class="td11">Repeat from step 2 </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-15-2"
| + | |
| − | class="td11">20 x </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-15-3"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-16-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-16-1"
| + | |
| − | class="td11">72 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-16-2"
| + | |
| − | class="td11">2 min </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-16-3"
| + | |
| − | class="td11"> </td>
| + | |
| − | </tr><tr
| + | |
| − | style="vertical-align:baseline;" id="TBL-2-17-"><td style="white-space:nowrap; text-align:left;" id="TBL-2-17-1"
| + | |
| − | class="td11">12 C </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-17-2"
| + | |
| − | class="td11">hold </td><td style="white-space:nowrap; text-align:left;" id="TBL-2-17-3"
| + | |
| − | class="td11"> </td></tr></table></div>
| + | |
| − | | + | |
| − | </div><hr class="endfloat" />
| + | |
| − | </div>
| + | |
| − | <h3 class="sectionHead"><span class="titlemark">2 </span> <a
| + | |
| − | id="x1-40002"></a>Results</h3>
| + | |
| − | <!--l. 40--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-50002"></a></span>
| + | |
| − | We cast a 1.5% TAE gel to visualize the results. We used 2 uL of NEB 50 bp
| + | |
| − | ladder. <hr class="figure"><div class="figure"
| + | |
| − | >
| + | |
| − | | + | |
| − | <a
| + | |
| − | id="x1-5001r1"></a>
| + | |
| − | | + | |
| − | | + | |
| − | <!--l. 44--><p class="noindent" ><img
| + | |
| − | src="https://static.igem.org/mediawiki/2015/0/07/4_13_2015.jpg" width="600" alt="PIC"
| + | |
| − | >
| + | |
| − | <br /> <div class="caption"
| + | |
| − | ><span class="id">Figure 1: </span><span
| + | |
| − | class="content">Annealing Temperature Test for Polymerase Chain Assembly of
| + | |
| − | MaSp2. Our target product is 170 bp, and is located just under the 200 bp
| + | |
| − | ladder band.</span></div><!--tex4ht:label?: x1-5001r1 -->
| + | |
| − | | + | |
| − | <!--l. 46--><p class="indent" > </div><hr class="endfigure">
| + | |
| − | <!--l. 47--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-60002"></a></span>
| + | |
| − | Our results indicate that 65 degrees is too high, and does not have good yield,
| + | |
| − | but 61.4 is too low and has non-specific amplification as shown by the smears.
| + | |
| − | The band at 50 bp is attributed to primer dimer formation.
| + | |
| − | <!--l. 49--><p class="noindent" ><span class="paragraphHead"><a
| + | |
| − | id="x1-70002"></a></span>
| + | |
| − | Based on our results, tomorrow, we will perform polymerase chain assembly
| + | |
| − | with annealing at 64 C and with 25 cycles.
| + | |
| − | </body>
| + | |
| − | </details>
| + | |
| | </details> | | </details> |
| − |
| |
| | </html> | | </html> |
| | | | |
Our goal is to be able to implement Iterative Capped Assmebly (ICA) for use in designing custom spider silk genes. The spider silk protein has a highly repetitive primary structure, and its properties can be changed by changing the sequence and identities of these repeats. We want to be able to fully control the primary sequence of our silk gene, and develop a quick, efficient method of customizing assembly of the spider-silk genes.
