|
|
| (25 intermediate revisions by the same user not shown) |
| Line 1: |
Line 1: |
| − | {{2015Top}}
| |
| − | <html>
| |
| | | | |
| − |
| |
| − | <!-- this is where the submenu is placed -->
| |
| − |
| |
| − |
| |
| − | <div id="leftColumn">
| |
| − | <h4><a href= "https://2015.igem.org/Troubleshooting">Troubleshooting</a></h4>
| |
| − | <ul>
| |
| − | <a href= "https://2015.igem.org/Troubleshooting#intro"> <li>Introduction</li></a>
| |
| − | <a href= "https://2015.igem.org/Troubleshooting#tips"> <li>General Tips and Tricks</li></a>
| |
| − | <ol><a href= "https://2015.igem.org/Troubleshooting/Transformation"> <li>Transformation</li></a>
| |
| − | <a href="https://2015.igem.org/Troubleshooting/Ligation"> <li>Ligation and Restriction Digest</li></ol>
| |
| − | <a href= "https://2015.igem.org/Troubleshooting#protocols"> <li>iGEM Protocols</li></a>
| |
| − | <a href= "https://2015.igem.org/Troubleshooting#answer"> <li>Answers</li></a>
| |
| − | <a href= "https://2015.igem.org/Troubleshooting#ask"> <li>Ask Your Questions</li></a>
| |
| − | </ul>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − |
| |
| − | <!-- this is where the content is placed --->
| |
| − | <div id="rightColumn">
| |
| − |
| |
| − | <!-- start of content ---------------------------------------->
| |
| − | <div class="alertMessage"> Page is under construction. <br>iGEM HQ is currently working on updating this information
| |
| − | for the iGEM 2015 competition. </div>
| |
| − |
| |
| − | <h2>Ligation and Restriction Digest Troubleshooting</h2>
| |
| − |
| |
| − | <p>
| |
| − | Now that you know your <a href"https://2015.igem.org/Troubleshooting/Transformation">transformation efficiency</a> and it's above 1 x 10<sup>8</sup> CFU/µg DNA, we can work on other possible problems if you're still not getting great results from your cloning. This page is focused on common problems researchers have with ligations and restriction digests.
| |
| − | <br><br>
| |
| − |
| |
| − | <b>Few or no colonies</b>
| |
| − | <p>
| |
| − | If you know your cells are working well, there are a few common ligation and digest problems that might be happening with your reaction.
| |
| − |
| |
| − | <ol><li><b>Uncut insert:</b> It's possible that your insert was not cut well during your digest.</li>
| |
| − | <li><b>Ligase didn't work:</b>
| |
| − |
| |
| − | </ol>
| |
| − | <br>
| |
| − | <b>Too many colonies</b>
| |
| − | <p>
| |
| − | Sometimes with ligation reactions you can end up with a lawn of bacterial growth where its impossible to select a single colony. While you may think this means your reaction worked really well, it actually indicates a problem with your restriction digest.
| |
| − |
| |
| − | <ol><li><b>Uncut backbone:</b> The most common cause of a lawn of bacteria after ligation is an uncut plasmid backbone. This makes your transformation in essence a plasmid transformation and you get far too many colonies on your plate.
| |
| − | <ol>To check for this, run a DNA agarose gel with your cut plasmid alongside a lane of uncut plasmid. Your uncut plasmid should appear to run smaller due to the supercoiled nature of uncut plasmid. Your cut plasmid should run higher since it's no longer supercoiled after being cut by the restriction enzyme(s).
| |
| − | <br><br>Also, for most BioBrick assemblies, when you cut open the backbone you are removing a piece of DNA from the cloning site. This will create two (or more) bands to appear on your gel.
| |
| − |
| |
| − |
| |
| − | <div class="clear"></div><div class="clear"></div>
| |
| − |
| |
| − |
| |
| − |
| |
| − | <!-- end of content ---->
| |
| − |
| |
| − | </div>
| |
| − | <div class="clear"></div>
| |
| − | </html> {{2015Bottom}}<html>
| |
| − | </div>
| |
| − | </html>
| |