Difference between revisions of "Team:Dundee/labjournal manu"
| Line 48: | Line 48: | ||
<div class="ribbon"> | <div class="ribbon"> | ||
<div class="rows"> | <div class="rows"> | ||
| + | |||
| + | |||
<!-- WEEK 1 --> | <!-- WEEK 1 --> | ||
| Line 112: | Line 114: | ||
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
<p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Click here to see our miniprep protocol.</a></p> | <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Click here to see our miniprep protocol.</a></p> | ||
| + | <p><a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol"> Click here to see our sequencing protocol.</a></p> | ||
</p> | </p> | ||
| − | <p><b>Results:</b> | + | <p><b>Results Miniprep: </b> |
| + | <table border="1" cellspacing="0" cellpadding="0"> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Sample Name</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Concentration</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="129" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Unit</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>Blank</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | - | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | - | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>BBa_K1058008</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 190.93 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | </tbody> | |
| + | </table> | ||
| + | </p> | ||
| − | <p><b> | + | <p><b>Results Sequencing: </b><a data-toggle="modal" data-target="#BBa_K1058008-figure1-modal" class="journal-protocol"> Figure 1 </a></p> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <p><b>Next Steps:</b> PCR of parts of <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a>.</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> | + | |
</div> | </div> | ||
| Line 162: | Line 189: | ||
| − | + | </div> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | ||
| − | + | ||
| − | |||
| − | + | <!-- WEEK 2 --> | |
| − | + | <div class="row"> | |
| − | + | <div class="border-week"> | |
| + | <div class="box"> | ||
| + | <div class="labtitle">29/06 - 05/07</div> | ||
| + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew2"></span> | ||
| + | <div class="box-content"> | ||
| + | <p class="journal-summary-heading">Summary</p> | ||
| + | |||
| − | + | <p class="journal-content"> During this week, <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> was disassembled into its constituent parts, pChr, and ChrB.</p> | |
| + | </div> | ||
| + | <div id="collapsiblew2" class="collapse week-content"> | ||
| − | + | <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div--> | |
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| Line 196: | Line 218: | ||
<div class="border-day"> | <div class="border-day"> | ||
<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 1</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew2-1"></span> |
| − | <div id=" | + | <div id="collapsiblew2-1" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To | + | <p><b>Aim of experiment: </b> To break down <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> into the promoter region pChr, and the repressor gene, ChrB, while adding standard prefix and suffix to each. Furthermore optimisation of first 15 codons of ChrB in order to increase the overall rate of transcription. Restriction digest of the respective ends and subsequent ligation of pChr and ChrB into pSB1C3 for submission to registry.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> Click here to see our PCR protocol.</a></p> |
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Click here to see our gel extraction protocol.</a></p> |
| + | <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Click here to see our restriction digest protocol.</a></p> | ||
| + | </p> | ||
| − | <p><b>Results:</b> | + | <p><b>Results:</b> |
| + | <p><a data-toggle="modal" data-target="#dundee15-chr-igem022-modal" class="journal-protocol"> Figure 2: Agarose gel after PCR of pChr and ChrB. </a></p> | ||
| + | <p>As can be seen on the image of the gel, PCR of ChrB was not successful. pChr was further processed by restriction digest with EcoRI and PstI</p></p> | ||
| − | |||
| − | + | <p><b>Next Steps:</b> Repeat of PCR of ChrB. Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> and confirmation of size and sequence.</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> and confirmation of size and sequence.</p> | + | |
</div> | </div> | ||
| Line 223: | Line 243: | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| Line 294: | Line 253: | ||
| − | <!-- WEEK | + | <!-- WEEK 3 --> |
<div class="row"> | <div class="row"> | ||
<div class="border-week"> | <div class="border-week"> | ||
<div class="box"> | <div class="box"> | ||
| − | <div class="labtitle"> | + | <div class="labtitle">06/07 - 12/07</div> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3"></span> |
<div class="box-content"> | <div class="box-content"> | ||
<p class="journal-summary-heading">Summary</p> | <p class="journal-summary-heading">Summary</p> | ||
| − | <p class="journal-content"> | + | <p class="journal-content">During this week ligations of pChr and ChrB into pSB1C3 were finalised and the constituent parts of our newly designed chromate sensing system were prepared for ligation and transformation.</p> |
</div> | </div> | ||
| − | <div id=" | + | <div id="collapsiblew3" class="collapse week-content"> |
<!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div--> | <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div--> | ||
| Line 317: | Line 276: | ||
<div class="box"> | <div class="box"> | ||
<span class="box-title">Day 1</span> <!--Title of collapsible box--> | <span class="box-title">Day 1</span> <!--Title of collapsible box--> | ||
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-1"></span> |
| − | <div id=" | + | <div id="collapsiblew3-1" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To | + | <p><b>Aim of experiment:</b> To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of pChr and ChrB into pSB1C3. Transformation of each into a non-pathogenic lab strain of E. coli. |
| + | <p>Overnight culture of strain, containing GFPmut2, as a fluorescent reporter for our Chromate sensing system</p></p> | ||
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> Click here to see our PCR protocol.</a> <p>The annealing temperature was lowered in order to increase the chances of accomodating a primer that is not 100% complementary. Furthermore different concentrations of DMSO were tested.</p></p> |
| − | <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Click here to see our | + | <p><a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Click here to see our gel extraction protocol.</a></p> |
| + | <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Click here to see our restriction digest protocol.</a></p> | ||
| + | <p><a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Click here to see our ligation protocol.</a> Ligations were made in 2:1 and 3:1 ratio.</p> | ||
| + | <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Click here to see our Overnight culture protocol.</a></p> | ||
| + | </p> | ||
| − | <p><b>Results:</b | + | <p><b>Results:</b> |
| − | + | <p><a data-toggle="modal" data-target="#dundee15-chr-igem023-modal" class="journal-protocol"> Figure 3: Agarose gel after PCR of ChrB. </a></p> | |
| − | + | <p>2 bands were extracted from the gel for further characterisation, the strongest band, hence called ChrBs, and the band directly above, hence called ChrBw.</p> | |
| − | + | <p>pChr, ChrBs, and ChrBw were ligated into pSB1C3.</p> | |
| − | <p | + | <p>pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 were transformed into E. coli MC1061.</p></p> |
| − | + | ||
| − | + | ||
| Line 348: | Line 310: | ||
<div class="box"> | <div class="box"> | ||
<span class="box-title">Day 2</span> <!--Title of collapsible box--> | <span class="box-title">Day 2</span> <!--Title of collapsible box--> | ||
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-2"></span> |
| − | <div id=" | + | <div id="collapsiblew3-2" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant MC1061, containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Overnight culture of pUniprom for subsequent ligation of ChrB</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep</a> for plasmid purification of overnight culture of GFPmut2.</p> |
| − | <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> | + | <p><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR</a> for amplification of GFPmut2 and ChrB.</p> |
| + | <p><a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel extraction</a> of PCR product pf GFPmut2.</p> | ||
| + | <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a> of MC1061 containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Furthermore of a strain containing the vector pUniprom.</p> | ||
| + | </p> | ||
| − | |||
| − | < | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem024-modal" class="journal-protocol"> Figure 5 </a> Since amplification of ChrB was unsuccessful, it will be repeated.</p> |
| − | |||
| − | + | <p><b>Next Steps:</b> Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3.</p> | |
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> | + | |
</div> | </div> | ||
| Line 379: | Line 339: | ||
<div class="box"> | <div class="box"> | ||
<span class="box-title">Day 3</span> <!--Title of collapsible box--> | <span class="box-title">Day 3</span> <!--Title of collapsible box--> | ||
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-3"></span> |
| − | <div id=" | + | <div id="collapsiblew3-3" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To | + | <p><b>Aim of experiment:</b> To purify overnight cultures for size and sequence confirmation. Repeat of PCR amplification of pChr and ChrB. Restriction digestes of all parts in order to produce compatible sticky ends.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep</a> of pChr-pSB1C3, ChrBs-pSB1C3, ChrBw-pSB1C3 and pUniprom.</p></p> |
| − | + | ||
| − | |||
| − | |||
| − | |||
| − | |||
| + | <p><b>Results Miniprep: </b> | ||
| + | <table border="1" cellspacing="0" cellpadding="0"> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Sample Name</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Concentration</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="129" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Unit</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| − | + | <strong>Blank</strong> | |
| − | + | </p> | |
| − | + | </td> | |
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | - | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | - | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom1</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 55.65 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| − | + | <strong>pUniprom2</strong> | |
| − | + | </p> | |
| − | + | </td> | |
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 64.20 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pChr1</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 116.58 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pChr2</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 110.13 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| + | <strong>pChr3</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 114.65 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pChr4</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 105.63 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>ChrBs1</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 151.18 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>ChrBs2</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 108.37 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>ChrBs3</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 143.13 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>ChrBs4</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 183.27 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| − | + | <strong>ChrBw1</strong> | |
| − | + | </p> | |
| − | + | </td> | |
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 116.26 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>ChrBw2</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 236.99 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| − | + | <strong>ChrBw3</strong> | |
| − | + | </p> | |
| − | + | </td> | |
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 219.21 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>ChrBw4</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 208.27 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | </tbody> | |
| + | </table> | ||
| + | </p> | ||
| − | |||
| − | |||
| − | |||
| − | + | <p><b>Protocols Used:</b> | |
| + | <p><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR</a> for amplification of pChr and ChrB.</p></p> | ||
| + | <p><a data-toggle="modal" data-target="#restriction_digest-modal" class="journal-protocol">Restriction digest</a> for confirmation of size of pChr and ChrB.</p></p> | ||
| − | < | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem026-modal" class="journal-protocol"> Figure 6 </a> Agarose gel for size confirmation.</p> |
| − | |||
| − | + | <p><b>Protocols Used:</b> | |
| + | <p><a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a> of pChr and ChrB that showed expectes sizes.</p></p> | ||
| + | |||
| + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-pChr1-modal" class="journal-protocol"> Figure 7 </a> Confirmed sequence of pChr1.</p> | ||
| + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-ChrBw4-modal" class="journal-protocol"> Figure 8 </a> Confirmed sequence of ChrBw4.</p> | ||
| + | |||
| + | <p><b>Protocols Used:</b> | ||
| + | <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction digest</a> of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.</p></p> | ||
| Line 471: | Line 676: | ||
<div class="border-day"> | <div class="border-day"> | ||
<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 4</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-4"></span> |
| − | <div id=" | + | <div id="collapsiblew3-4" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To prepare | + | <p><b>Aim of experiment:</b> To prepare pUniprom for insertion of ChrB.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> of pUniprom with BamHI and HindIII.</p> |
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#gel-extraction-modal" class="journal-protocol">Gel extraction</a> of digested pUniprom</p> |
| + | </p> | ||
| − | <p><b>Results:</b> | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem034-modal" class="journal-protocol"> Figure 9 </a></p> |
| − | |||
| − | <p><b> | + | <p><b>Next Steps:</b> Ligation of ChrB into pUniprom.</p> |
| − | |||
| + | <p><b>Aim of experiment:</b> To amplify ChrB version with first 15 codons optimised for insertion into pUniprom.</p> | ||
| − | <p><b> | + | <p><b>Protocols Used:</b> |
| + | <p><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol">PCR</a> of optimised ChrB from the sequence confirmed ChrBw4-pSB1C3, adding restriction sites compatible to pUniprom.</p> | ||
| + | <p><a data-toggle="modal" data-target="#gelextraction" class="journal-protocol">Gel extraction</a> of optimised ChrBafter PCR.</p> | ||
| + | </p> | ||
| + | |||
| + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem034-modal" class="journal-protocol"> Figure 10 </a></p> | ||
| + | |||
| + | |||
| + | <p><b>Next Steps:</b> Ligation of ChrB and codon optimised ChrB into pUniprom.</p> | ||
</div> | </div> | ||
| Line 502: | Line 715: | ||
<div class="border-day"> | <div class="border-day"> | ||
<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 5</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-5"></span> |
| − | <div id=" | + | <div id="collapsiblew3-5" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> Ligation of ChrB and optimised ChrB into pUniprom. Ligation of pChr and GFPmut2 into pSB1C3</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol">Ligation</a> of ChrB and optimised ChrB into pUniprom via BamHI and HindIII restriction sites. Ligation of pChr to GFPmut2 via Spe/Xba link and ligation of pChrGFP construct into pSB1C3 EcoRI/PstI restriction sites.</p> |
| − | + | </p> | |
<p><b>Results:</b> N/A</p> | <p><b>Results:</b> N/A</p> | ||
| − | |||
| − | + | <p><b>Next Steps:</b>Transformation of abovementioned ligations.</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> | + | |
</div> | </div> | ||
| Line 538: | Line 745: | ||
| − | <!-- WEEK | + | <!-- WEEK 4 --> |
<div class="row"> | <div class="row"> | ||
<div class="border-week"> | <div class="border-week"> | ||
<div class="box"> | <div class="box"> | ||
| − | <div class="labtitle"> | + | <div class="labtitle">13/07 - 19/07</div> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4"></span> |
<div class="box-content"> | <div class="box-content"> | ||
<p class="journal-summary-heading">Summary</p> | <p class="journal-summary-heading">Summary</p> | ||
| − | <p class="journal-content"> | + | <p class="journal-content">During this week successful transformations were identifeid and propagated.></p> |
</div> | </div> | ||
| − | <div id=" | + | <div id="collapsiblew4" class="collapse week-content"> |
<!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div--> | <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div--> | ||
| Line 561: | Line 768: | ||
<div class="box"> | <div class="box"> | ||
<span class="box-title">Day 1</span> <!--Title of collapsible box--> | <span class="box-title">Day 1</span> <!--Title of collapsible box--> | ||
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-1"></span> |
| − | <div id=" | + | <div id="collapsiblew4-1" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> Transform recombinant vectors into E. coli chassis.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation</a> of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.</p> |
| − | + | </p> | |
| − | |||
| − | < | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-first-transformation-modal" class="journal-protocol"> Figure 11 </a></p> |
| − | |||
| − | + | <p><b>Aim of experiment:</b> Backup of digested pSB1C3 for the case that a ligation with 2 inserts does not work.</p> | |
| − | + | ||
| − | + | ||
| − | <p><b> | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> of pSB1C3 with EcoRI/SpeI for subsequent insertion of pChr.</p> |
| − | + | </p> | |
| − | |||
| − | < | + | <p><b>Results:</b>N/A</p> |
| − | + | <p><b>Next Steps:</b> Store digested plasmid at -20<sup>o</sup>C.</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> | + | |
</div> | </div> | ||
| Line 622: | Line 802: | ||
<div class="border-day"> | <div class="border-day"> | ||
<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 2</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-2"></span> |
| − | <div id=" | + | <div id="collapsiblew4-2" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> Purification of recombinant vectors for sequence and size confirmation.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a> of JM110 with ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.</p> |
| − | + | <p><a data-toggle="modal" data-target="#plating-modal" class="journal-protocol"> Patch plating</a> of the same colonies.</p> | |
| + | </p> | ||
<p><b>Results:</b> N/A</p> | <p><b>Results:</b> N/A</p> | ||
| Line 642: | Line 823: | ||
| − | <p><b>Next Steps:</b> Purification of the | + | <p><b>Next Steps:</b> Purification of the recombinant plasmids for confirmation of size and sequence.</p> |
</div> | </div> | ||
| Line 653: | Line 834: | ||
<div class="border-day"> | <div class="border-day"> | ||
<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 3</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-3"></span> |
| − | <div id=" | + | <div id="collapsiblew4-3" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> Purification of recombinant plasmids for confirmation of size and sequence.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Miniprep</a> of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.</p> |
| − | + | </p> | |
| − | <p><b>Results:</b | + | <p><b>Results:</b> |
| − | |||
| − | + | <table border="1" cellspacing="0" cellpadding="0"> | |
| + | <tbody> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Sample Name</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Concentration</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="129" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Unit</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>Blank</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | - | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | - | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pSB1C3-pChr-GFPmut2_1</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 382.32 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| − | + | <strong>pSB1C3-pChr-GFPmut2_2</strong> | |
| − | + | </p> | |
| − | + | </td> | |
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 355.31 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pSB1C3-pChr-GFPmut2_3</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 209.85 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| − | + | <strong>pSB1C3-pChr-GFPmut2_4</strong> | |
| − | + | </p> | |
| − | + | </td> | |
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 217.57 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom-ChrB-opt_1</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 150.51 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom-ChrB-opt_2</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 214.17 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| + | <strong>pUniprom-ChrB-opt_3</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 140.85 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom-ChrB-opt_4</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 115.85 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom-ChrB_1</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 134.72 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom-ChrB_2</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 111.41 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom-ChrB_3</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 111.31 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom-ChrB_4</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 138.9 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | </tbody> | |
| − | + | </table> | |
| − | + | </p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <p><b>Protocols Used:</b> | |
| − | + | <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> of ChrB-pUniprom and optimised ChrB-pUniprom with BamHI and HindIII. Restriction digest of pChr-GFPmut2-pSB1C3 with EcoRI/PstI.</p> | |
| − | + | </p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem036-modal" class="journal-protocol"> Figure 1 </a></p> | ||
| − | |||
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#colony-modal" class="journal-protocol">Colony PCR</a> of pUniprom-ChrB-opt for elucidation of insert size or presence of insert.</p> |
| − | + | </p> | |
| − | <p><b>Results:</b> | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem037-modal" class="journal-protocol"> Figure 13 </a> Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.</p> |
| − | |||
| − | + | <p><b>Next Steps:</b> Confirmation of insert size and sequence of ChrB-opt.</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> | + | |
</div> | </div> | ||
| Line 746: | Line 1,132: | ||
<div class="border-day"> | <div class="border-day"> | ||
<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 4</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-4"></span> |
| − | <div id=" | + | <div id="collapsiblew4-4" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To prepare | + | <p><b>Aim of experiment:</b> To prepare ChrB-opt colonies 2, 4, 9, and 12 for sequencing.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | |||
<p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Click here to see our overnight culture protocol.</a></p></p> | <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Click here to see our overnight culture protocol.</a></p></p> | ||
| − | <p><b>Results:</b> N/A</p> | + | <p><b>Results:</b>N/A</p> |
<!-- For images in the results use the following line of code: | <!-- For images in the results use the following line of code: | ||
| Line 766: | Line 1,151: | ||
| − | <p><b>Next Steps:</b> Purification of | + | <p><b>Next Steps:</b> Purification of recombinant plasmids.</p> |
</div> | </div> | ||
| Line 772: | Line 1,157: | ||
</div> | </div> | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| Line 804: | Line 1,162: | ||
<div class="border-day"> | <div class="border-day"> | ||
<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 5</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-5"></span> |
| − | <div id=" | + | <div id="collapsiblew4-5" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To | + | <p><b>Aim of experiment:</b> To purify recombinant plasmids for sequence confirmation.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep</a> of ChrB-opt 2, 4, 9, 12.</p> |
| − | + | </p> | |
| − | <p><b>Results:</b | + | <p><b>Results:</b> |
| − | |||
| − | + | <table border="1" cellspacing="0" cellpadding="0"> | |
| + | <tbody> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Sample Name</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Concentration</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="129" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Unit</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>Blank</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | - | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | - | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom-ChrB-opt_2</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 255.35 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| − | + | <strong>pUniprom-ChrB-opt_4</strong> | |
| − | + | </p> | |
| − | + | </td> | |
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 146.77 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="140" valign="top"> | ||
| + | <p> | ||
| + | <strong>pUniprom-ChrB-opt_9</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 211.91 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td width="140" valign="top"> | |
| − | + | <p> | |
| − | + | <strong>pUniprom-ChrB-opt_12</strong> | |
| − | + | </p> | |
| − | + | </td> | |
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | 181.08 | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="111" valign="top"> | ||
| + | <p align="center"> | ||
| + | ng/µl | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | </p> | ||
| − | |||
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | <p><a data-toggle="modal" data-target="# | + | <p><a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a> of ChrB-opt 2, 4, 9, 12.</p> |
| − | + | </p> | |
| − | <p><b>Results:</b> | + | <p><b>Results:</b> ChrB showed HindIII restriction sites and was hence cleaved into partial inserts.</p> |
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<!-- For images in the results use the following line of code: | <!-- For images in the results use the following line of code: | ||
| Line 2,524: | Line 2,881: | ||
<!-- POPUP PROTOCOL MODAL SECTION --> | <!-- POPUP PROTOCOL MODAL SECTION --> | ||
| + | |||
| + | |||
| + | <!-- Plating Modal --> | ||
| + | <div class="modal fade" id="plating-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Plating Protocol</div> | ||
| + | </div> | ||
| + | <div class="modal-body"><p> | ||
| + | 1. Agar plates with required antibiotic were taken out of 4<sup>o</sup>C to warm to room temperature. | ||
| + | </p> | ||
| + | <p> | ||
| + | 2. Cells or colonies were picked up from the source with a toothpick under sterile conditions. | ||
| + | </p> | ||
| + | <p> | ||
| + | 3. Fresh agar plates were inoculated with cells on the toothpick. | ||
| + | </p> | ||
| + | <p> | ||
| + | 4. Agar plates were incubated at 4<sup>o</sup>C over night. | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <!-- Sequencing Modal --> | ||
| + | <div class="modal fade" id="sequencing-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Sequencing Protocol</div> | ||
| + | </div> | ||
| + | <div class="modal-body"><p> | ||
| + | For sequencing a sample, the following ingredients were added to a microcentrifuge tube: | ||
| + | <ul> | ||
| + | <li>Volume of sample equivalent to 550ng DNA</li> | ||
| + | <li>2µl of 3mM primer</li> | ||
| + | <li>Remaining volume of water for a total volume of 30µl</li> | ||
| + | </ul> | ||
| + | </p> | ||
| + | <p> | ||
| + | The samples were submitted to <a href="https://www.dnaseq.co.uk/" target="_blank">DNA sequencing and services</a>. | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <!-- Overnightculture Modal --> | ||
| + | <div class="modal fade" id="overnightculture-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Overnight Culture Protocol</div> | ||
| + | </div> | ||
| + | <div class="modal-body"><p> | ||
| + | 1. Agar plates with required antibiotic were taken out of 4<sup>o</sup>C to warm to room temperature. | ||
| + | </p> | ||
| + | <p> | ||
| + | 2. Cells or colonies were picked up from the source with a toothpick under sterile conditions. | ||
| + | </p> | ||
| + | <p> | ||
| + | 3. 5ml of LB medium, supplemented with the required antibiotic, were inoculated with the toothpick. | ||
| + | </p> | ||
| + | <p> | ||
| + | 4. Tubes were incubated at 37<sup>o</sup>C in a rotary incubator at 200rpm over night. | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
<!-- PCR Modal --> | <!-- PCR Modal --> | ||
<div class="modal fade" id="PCR-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | <div class="modal fade" id="PCR-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| Line 2,666: | Line 3,111: | ||
| + | <!-- Colony PCR Modal --> | ||
| + | <div class="modal fade" id="colony-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Colony PCR Protocol</div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | |||
| + | <p> | ||
| + | The PCR reaction mixture were set up in multiples of the volumes in table below. The length of all sequences used is stated in the <strong>Sequences</strong> section. | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | The DNA template is made in the following way: | ||
| + | <ol> | ||
| + | <li>Agar plates with required antibiotic were taken out of 4<sup>o</sup>C to warm to room temperature.</li> | ||
| + | <li>A numbered grid according to the desired number of PCR reactions is drawn on the back of the plate.</li> | ||
| + | <li>Cells or colonies were picked up from the source with a toothpick under sterile conditions.</li> | ||
| + | <li>Each colony is transferred to a 30µl aliquot of sterile water.</li> | ||
| + | <li>With the same toothpick the grid with the respctive number on the plate was inoculated.</li> | ||
| + | <li><ul> | ||
| + | <li>The plate was incubated at 37<sup>o</sup>C overnight.</li> | ||
| + | <li>The inoculated water-aliquots were boiled for 10min, then centrifuged at 13300rpm for 10min.</li> | ||
| + | </ul> | ||
| + | <li>The supernatant of this preparation is the DNA fraction for the subsequent PCR reaction.</li> | ||
| + | </ol> | ||
| + | </p> | ||
| + | |||
| + | <table border="1" cellspacing="0" cellpadding="0"> | ||
| + | |||
| + | <tbody color="black"> | ||
| + | |||
| + | <tr> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Reactants</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | <strong>Volume (µl)</strong> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | DNA | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | 5 | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | DMSO | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | 1 | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | Forward Primer | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | 0.5 | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | Reverse Primer | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | 0.5 | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | +Mg Buffer | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | 2 | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | dNTP | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | 0.2 | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | Taq-polymerase | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | 0.2 | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | H<sub>2</sub>O | ||
| + | </p> | ||
| + | </td> | ||
| + | <td width="312" valign="top"> | ||
| + | <p align="center"> | ||
| + | 10.6 | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | </tbody> | ||
| + | </table> | ||
| + | |||
| + | <p>15µl of the reaction mixture was aliquoted into separate PCR tubes. 5µl of DNA fraction was added to each respective tube in order to have a total reaction volume of 20µl per tube.</p> | ||
| + | |||
| + | <p> | ||
| + | **PCR Programme** | ||
| + | </p> | ||
| + | <p> | ||
| + | Elongation time was altered according to the length of the sequences. | ||
| + | </p> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <!-- Miniprep Modal --> | ||
| + | <div class="modal fade" id="miniprep-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Miniprep Protocol</div> | ||
| + | </div> | ||
| + | <div class="modal-body"><p> | ||
| + | 1. 5ml bacterial overnight culture was pelleted by stepwise centrifugation in 1.5ml microcentrifuge tubes at 13300rpm for 3 minutes at room temperature. | ||
| + | </p> | ||
| + | <p> | ||
| + | 2. Pelleted bacterial cells were resuspended in 250µl of Buffer P1. | ||
| + | </p> | ||
| + | <p> | ||
| + | 3. 250µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution became clear. | ||
| + | </p> | ||
| + | <p> | ||
| + | 4. 350µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times. | ||
| + | </p> | ||
| + | <p> | ||
| + | 5. The mixture was centrifuged for 10 minutes at 13300rpm in a microcentrifuge. | ||
| + | </p> | ||
| + | <p> | ||
| + | 6. The supernatant was applied to a QIAprep spin column by pipetting. | ||
| + | </p> | ||
| + | <p> | ||
| + | 7. 500µl of Buffer PB was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded. | ||
| + | </p> | ||
| + | <p> | ||
| + | 8. 750µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded. | ||
| + | </p> | ||
| + | <p> | ||
| + | 9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer. | ||
| + | </p> | ||
| + | <p> | ||
| + | 10. The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H2O to the QIAprep spin column and centrifuging for 1 minute. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <!-- Gel extraction Modal --> | ||
| + | <div class="modal fade" id="gelextraction-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Gel Extraction Protocol</div> | ||
| + | </div> | ||
| + | <div class="modal-body"><p> | ||
| + | 1. The desired DNA fragment was excised from the agarose gel with a clean, sharp scalpel. | ||
| + | </p> | ||
| + | <p> | ||
| + | 2. The gel slice was transferred into a microcentrifuge tube. 700µl of Buffer QG was added. | ||
| + | </p> | ||
| + | <p> | ||
| + | 3. The gel slice was incubated in a water bath at 50°C for 10 min (or until the gel slice has completely dissolved). The tube was vortexed every 2–3 min to help dissolve gel. | ||
| + | </p> | ||
| + | <p> | ||
| + | 4. After the gel slice had been dissolved completely, the color of the mixture was checked that it was yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture was orange or violet, 10 μl 3 M sodium acetate, pH 5.0 would be added, and mixed. The color of the mixture would turn yellow. | ||
| + | </p> | ||
| + | <p> | ||
| + | 5. 230µl of isopropanol was added to the sample and mixed. | ||
| + | </p> | ||
| + | <p> | ||
| + | 6. A QIAquick spin column was placed in a 2ml collection tube. The sample was applied to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded. | ||
| + | </p> | ||
| + | <p> | ||
| + | 7. 500µl of QG buffer was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded. | ||
| + | </p> | ||
| + | <p> | ||
| + | 8. 750µl of Buffer PE was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded. | ||
| + | </p> | ||
| + | <p> | ||
| + | 9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer. | ||
| + | </p> | ||
| + | <p> | ||
| + | 10. The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H2O to the QIAprep spin column and centrifuging for 1 minute. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| Line 2,848: | Line 3,559: | ||
The digests were incubated at 37<sup>o</sup>C for 3 hours. | The digests were incubated at 37<sup>o</sup>C for 3 hours. | ||
<br/> | <br/> | ||
| − | Note that when performing plasmid digestions, 2.5µl of alkaline phosphatase and 2.5µl of its buffer | + | Note that when performing plasmid digestions, 2.5µl of alkaline phosphatase and 2.5µl of its buffer were added at the 2 hour and 2.5 hour mark. |
</p> | </p> | ||
| Line 2,859: | Line 3,570: | ||
</div> | </div> | ||
</div> | </div> | ||
| + | |||
<!-- Ligation Modal --> | <!-- Ligation Modal --> | ||
| Line 3,098: | Line 3,810: | ||
</p> | </p> | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
</div> | </div> | ||
<div class="modal-footer"> | <div class="modal-footer"> | ||
| Line 3,231: | Line 3,885: | ||
</p> | </p> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <!--My Image modals --> | ||
| + | |||
| + | <div class="modal fade" id="BBa_K1058008-figure1-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 1: Alignment of <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a>. Sequences from top to bottom: sequencing result from forward primer; expected sequence; reverse complement of sequencing result from reverse primer; sequencing result from intermediate primer. Colour code: Green = Prefix/Suffix; Pink = Annealing site of intermediate primer; Yellow = Mismatch in one sequence; Red = In frame stop codons. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/1/1d/Cleaned_alignment2.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-igem022-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 2: 1% agarose gel after PCR of <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> with primers specific to the promoter region, pChr, and the repressor region, ChrB. Left: pChr (expected size 169bp); Middle: 1kb+ ladder, Right: ChrB (expected size 939bp). | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/c/c0/Dundee15_chr_igem022.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-igem023-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 3: 1% agarose gel after PCR of <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> with primers specific to the repressor region, ChrB. Left: 1.5µl DMSO; Middle: 1kb+ ladder, Right: 2.5µl DMSO. Expected size for each: 939bp. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/7/7c/Dundee15_chr_igem023.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-igem024-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 4: 1% agarose gel after PCR of GFPmut2 with corresponding primers primers. Left: GFPmut2 (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of ChrB, using primers to add a BamHI restriction site in front of it and a His-Tag and a HindIII restriction site at the end. (expected size of ChrB: 939bp). | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/3/30/Dundee15_chr_igem024.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-igem026-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 5: 1% agarose gel after restriction digest of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 for confirmation of size. Ladder: 1kb+. Top: 1-4: pChr 1-4; ChrBs 1-2. Bottom: ChrBs 3-4; ChrBw 1-4. Expected sizes: pChr: 169bp; ChrB: 939bp. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/f/ff/Dundee15_chr_igem026.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-pChr1-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 6: 1% agarose gel after PCR of GFPmut2 with corresponding primers primers. Left: GFPmut2 (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of ChrB, using primers to add a BamHI restriction site in front of it and a His-Tag and a HindIII restriction site at the end. (expected size of ChrB: 939bp). Colours: Green: Prefix and Suffix; Red: In frame stop codons. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/4/41/Dundee15_chr_pChr1.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-ChrBw4-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 7: 1% agarose gel after PCR of GFPmut2 with corresponding primers primers. Left: GFPmut2 (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of ChrB, using primers to add a BamHI restriction site in front of it and a His-Tag and a HindIII restriction site at the end. (expected size of ChrB: 939bp). Colours: Green: Prefix and Suffix; Red: In frame stop codons; Blue: First 15 codons optimised for expression in E. coli. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/7/7a/Dundee15_chr_ChrBw4.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-igem034-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 8: 1% agarose gel after restriction digest of pUniprom (Expected size 2454bp). | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/0/0b/Dundee15_chr_igem034.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-igem035-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 10: 1% agarose gel after PCR of ChrBw4 (Expected size 939bp). | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/6/6f/Dundee15_chr_igem035.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-first-transformation-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 11: Plates after transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/2/29/Dundee15_chr_first-transformation.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-igem036-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 12: 1% agarose gel after restriction digest ofChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 for confirmation of size. Ladder: 1kb+. Top: 1-4: pSB1C3-pChr-GFP 1-4; pUniprom-ChrB-opt 1-2. Bottom: pUniprom-ChrB-opt 3-4; pUniprom-ChrB 1-4. Expected sizes: pChr-GFP: 909bp; ChrB and Chrb-opt: 939bp. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/4/41/Dundee15_chr_igem036.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15-chr-igem037-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header"> | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 13: 1% agarose gel after colony PCR of pUniprom-ChrB-opt. Top: colonies 1-11; Bottom: colonies 12-22. Ladder: 1kb+ Expected size: 939bp. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/6/6b/Dundee15_chr_igem037.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
</div> | </div> | ||
<div class="modal-footer"> | <div class="modal-footer"> | ||
| Line 3,252: | Line 4,172: | ||
<img src="https://static.igem.org/mediawiki/2015/8/85/Dundee15-LbpAPCR-pSB1C3.jpg" style="width:400px;height:auto">' | <img src="https://static.igem.org/mediawiki/2015/8/85/Dundee15-LbpAPCR-pSB1C3.jpg" style="width:400px;height:auto">' | ||
| − | + | !--insert link of uploaded files here-- | |
</div> | </div> | ||
Revision as of 19:16, 8 August 2015
LABJOURNAL
BioSpray
Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To purify BBa_K1058008 for sequence confirmation.
Protocols Used:
Click here to see our miniprep protocol.
Click here to see our sequencing protocol.
Results Miniprep:
|
Sample Name |
Concentration |
Unit |
|
Blank |
- |
- |
|
BBa_K1058008 |
190.93 |
ng/µl |
Results Sequencing: Figure 1
Next Steps: PCR of parts of BBa_K1058008.
Summary
During this week, BBa_K1058008 was disassembled into its constituent parts, pChr, and ChrB.
Aim of experiment: To break down BBa_K1058008 into the promoter region pChr, and the repressor gene, ChrB, while adding standard prefix and suffix to each. Furthermore optimisation of first 15 codons of ChrB in order to increase the overall rate of transcription. Restriction digest of the respective ends and subsequent ligation of pChr and ChrB into pSB1C3 for submission to registry.
Protocols Used:
Click here to see our PCR protocol.
Click here to see our gel extraction protocol.
Click here to see our restriction digest protocol.
Results:
Figure 2: Agarose gel after PCR of pChr and ChrB.
As can be seen on the image of the gel, PCR of ChrB was not successful. pChr was further processed by restriction digest with EcoRI and PstI
Next Steps: Repeat of PCR of ChrB. Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
During this week ligations of pChr and ChrB into pSB1C3 were finalised and the constituent parts of our newly designed chromate sensing system were prepared for ligation and transformation.
Aim of experiment: To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of pChr and ChrB into pSB1C3. Transformation of each into a non-pathogenic lab strain of E. coli.
Overnight culture of strain, containing GFPmut2, as a fluorescent reporter for our Chromate sensing system
Protocols Used:
Click here to see our PCR protocol.
The annealing temperature was lowered in order to increase the chances of accomodating a primer that is not 100% complementary. Furthermore different concentrations of DMSO were tested.
Click here to see our gel extraction protocol.
Click here to see our restriction digest protocol.
Click here to see our ligation protocol. Ligations were made in 2:1 and 3:1 ratio.
Click here to see our Overnight culture protocol.
Results:
Figure 3: Agarose gel after PCR of ChrB.
2 bands were extracted from the gel for further characterisation, the strongest band, hence called ChrBs, and the band directly above, hence called ChrBw.
pChr, ChrBs, and ChrBw were ligated into pSB1C3.
pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 were transformed into E. coli MC1061.
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant MC1061, containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Overnight culture of pUniprom for subsequent ligation of ChrB
Protocols Used:
Miniprep for plasmid purification of overnight culture of GFPmut2.
PCR for amplification of GFPmut2 and ChrB.
Gel extraction of PCR product pf GFPmut2.
Overnight culture of MC1061 containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Furthermore of a strain containing the vector pUniprom.
Results: Figure 5 Since amplification of ChrB was unsuccessful, it will be repeated.
Next Steps: Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3.
Aim of experiment: To purify overnight cultures for size and sequence confirmation. Repeat of PCR amplification of pChr and ChrB. Restriction digestes of all parts in order to produce compatible sticky ends.
Protocols Used:
Miniprep of pChr-pSB1C3, ChrBs-pSB1C3, ChrBw-pSB1C3 and pUniprom.
Results Miniprep:
|
Sample Name |
Concentration |
Unit |
|
Blank |
- |
- |
|
pUniprom1 |
55.65 |
ng/µl |
|
pUniprom2 |
64.20 |
ng/µl |
|
pChr1 |
116.58 |
ng/µl |
|
pChr2 |
110.13 |
ng/µl |
|
pChr3 |
114.65 |
ng/µl |
|
pChr4 |
105.63 |
ng/µl |
|
ChrBs1 |
151.18 |
ng/µl |
|
ChrBs2 |
108.37 |
ng/µl |
|
ChrBs3 |
143.13 |
ng/µl |
|
ChrBs4 |
183.27 |
ng/µl |
|
ChrBw1 |
116.26 |
ng/µl |
|
ChrBw2 |
236.99 |
ng/µl |
|
ChrBw3 |
219.21 |
ng/µl |
|
ChrBw4 |
208.27 |
ng/µl |
Protocols Used:
PCR for amplification of pChr and ChrB.
Restriction digest for confirmation of size of pChr and ChrB.
Results: Figure 6 Agarose gel for size confirmation.
Protocols Used:
Sequencing of pChr and ChrB that showed expectes sizes.
Results: Figure 7 Confirmed sequence of pChr1.
Results: Figure 8 Confirmed sequence of ChrBw4.
Protocols Used:
Restriction digest of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare pUniprom for insertion of ChrB.
Protocols Used:
Restriction digest of pUniprom with BamHI and HindIII.
Gel extraction of digested pUniprom
Results: Figure 9
Next Steps: Ligation of ChrB into pUniprom.
Aim of experiment: To amplify ChrB version with first 15 codons optimised for insertion into pUniprom.
Protocols Used:
PCR of optimised ChrB from the sequence confirmed ChrBw4-pSB1C3, adding restriction sites compatible to pUniprom.
Gel extraction of optimised ChrBafter PCR.
Results: Figure 10
Next Steps: Ligation of ChrB and codon optimised ChrB into pUniprom.
Aim of experiment: Ligation of ChrB and optimised ChrB into pUniprom. Ligation of pChr and GFPmut2 into pSB1C3
Protocols Used:
Ligation of ChrB and optimised ChrB into pUniprom via BamHI and HindIII restriction sites. Ligation of pChr to GFPmut2 via Spe/Xba link and ligation of pChrGFP construct into pSB1C3 EcoRI/PstI restriction sites.
Results: N/A
Next Steps:Transformation of abovementioned ligations.
Summary
During this week successful transformations were identifeid and propagated.>
Aim of experiment: Transform recombinant vectors into E. coli chassis.
Protocols Used:
Transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.
Results: Figure 11
Aim of experiment: Backup of digested pSB1C3 for the case that a ligation with 2 inserts does not work.
Protocols Used:
Restriction digest of pSB1C3 with EcoRI/SpeI for subsequent insertion of pChr.
Results:N/A
Next Steps: Store digested plasmid at -20oC.
Aim of experiment: Purification of recombinant vectors for sequence and size confirmation.
Protocols Used:
Overnight culture of JM110 with ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.
Patch plating of the same colonies.
Results: N/A
Next Steps: Purification of the recombinant plasmids for confirmation of size and sequence.
Aim of experiment: Purification of recombinant plasmids for confirmation of size and sequence.
Protocols Used:
Miniprep of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.
Results:
|
Sample Name |
Concentration |
Unit |
|
Blank |
- |
- |
|
pSB1C3-pChr-GFPmut2_1 |
382.32 |
ng/µl |
|
pSB1C3-pChr-GFPmut2_2 |
355.31 |
ng/µl |
|
pSB1C3-pChr-GFPmut2_3 |
209.85 |
ng/µl |
|
pSB1C3-pChr-GFPmut2_4 |
217.57 |
ng/µl |
|
pUniprom-ChrB-opt_1 |
150.51 |
ng/µl |
|
pUniprom-ChrB-opt_2 |
214.17 |
ng/µl |
|
pUniprom-ChrB-opt_3 |
140.85 |
ng/µl |
|
pUniprom-ChrB-opt_4 |
115.85 |
ng/µl |
|
pUniprom-ChrB_1 |
134.72 |
ng/µl |
|
pUniprom-ChrB_2 |
111.41 |
ng/µl |
|
pUniprom-ChrB_3 |
111.31 |
ng/µl |
|
pUniprom-ChrB_4 |
138.9 |
ng/µl |
Protocols Used:
Restriction digest of ChrB-pUniprom and optimised ChrB-pUniprom with BamHI and HindIII. Restriction digest of pChr-GFPmut2-pSB1C3 with EcoRI/PstI.
Results: Figure 1
Protocols Used:
Colony PCR of pUniprom-ChrB-opt for elucidation of insert size or presence of insert.
Results: Figure 13 Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.
Next Steps: Confirmation of insert size and sequence of ChrB-opt.
Aim of experiment: To prepare ChrB-opt colonies 2, 4, 9, and 12 for sequencing.
Protocols Used:
Click here to see our overnight culture protocol.
Results:N/A
Next Steps: Purification of recombinant plasmids.
Aim of experiment: To purify recombinant plasmids for sequence confirmation.
Protocols Used:
Miniprep of ChrB-opt 2, 4, 9, 12.
Results:
|
Sample Name |
Concentration |
Unit |
|
Blank |
- |
- |
|
pUniprom-ChrB-opt_2 |
255.35 |
ng/µl |
|
pUniprom-ChrB-opt_4 |
146.77 |
ng/µl |
|
pUniprom-ChrB-opt_9 |
211.91 |
ng/µl |
|
pUniprom-ChrB-opt_12 |
181.08 |
ng/µl |
Protocols Used:
Sequencing of ChrB-opt 2, 4, 9, 12.
Results: ChrB showed HindIII restriction sites and was hence cleaved into partial inserts.
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Restriction digests were set up according to the table below. The appropriate reaction and restriction enzymes that were used is stated in the Journal section.
|
Post Plasmid Purification |
Post PCR |
gBlock |
|
|
DNA |
1mg |
50µl |
100ng |
|
Cutsmart Buffer (µl) |
6 |
6 |
6 |
|
Water (µl) |
As required |
1 |
As required |
|
Enzyme 1 (µl) |
1.5 |
1.5 |
1.5 |
|
Enzyme 2 (µl) |
1.5 |
1.5 |
1.5 |
|
Total Volume (µl) |
30 |
60 |
30 |
The digests were incubated at 37oC for 3 hours.
Note that when performing plasmid digestions, 2.5µl of alkaline phosphatase and 2.5µl of its buffer were added at the 2 hour and 2.5 hour mark.
Ligation reactions were set up according to the table below. The vector to insert ratio is stated for each specific reaction in the Journal section.
|
Vector (µl) |
2:1 (Vector: Insert) [µl] |
3:1 (Vector: Insert) [µl] |
4:1 (Vector: Insert) [µl] |
|
|
T4 DNA Ligase |
1 |
1 |
1 |
1 |
|
Ligase Buffer |
1 |
1 |
1 |
1 |
|
Vector |
1 |
1 |
1 |
1 |
|
Insert |
- |
2 |
3 |
4 |
|
H20 |
7 |
5 |
4 |
3 |
The reactions were incubated at room temperature for at least 3 hours.
'
'
'
'
'
'
'
'
'
'
'
'