Team:StanfordBrownSpelman/Lab Techniques8
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- | <b>Designing Primers</b | + | <b>Designing Primers</b> |
<br />Choose a forward and reverse primer from a location in the gene or plasmid that is sure to include the portion desired for amplification or sequencing | <br />Choose a forward and reverse primer from a location in the gene or plasmid that is sure to include the portion desired for amplification or sequencing | ||
For sequencing, it is desirable if possible to have primers that fall 50-150bp outside your desired region, to ensure that accurate reading occurs for the whole gene (often the first and last ~100bp in the read are very inaccurate). For PCR remember that the sequence portion corresponding to the primers themselves will be amplified also | For sequencing, it is desirable if possible to have primers that fall 50-150bp outside your desired region, to ensure that accurate reading occurs for the whole gene (often the first and last ~100bp in the read are very inaccurate). For PCR remember that the sequence portion corresponding to the primers themselves will be amplified also | ||
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Special BioBrick considerations | Special BioBrick considerations | ||
- | <br /><br /><b>Ordering Primers</b> | + | <br /><br /><b>Ordering Primers</b><br> |
We order our primers from <a href="http://elimbio.com/" target="_blank">ELIM Biopharm.</a> The rest is fairly self-explanatory, but we'll do a walk through when you get here | We order our primers from <a href="http://elimbio.com/" target="_blank">ELIM Biopharm.</a> The rest is fairly self-explanatory, but we'll do a walk through when you get here | ||
Primers <36bp ordered before 5pm will arrive the next day. Delivery is around 2:15pm. | Primers <36bp ordered before 5pm will arrive the next day. Delivery is around 2:15pm. | ||
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<h6 class="subindent"> | <h6 class="subindent"> | ||
<br /><b>Example:</b> | <br /><b>Example:</b> | ||
- | 10μL primer stock | + | 10μL primer stock<br> |
190μL qH2O or TE buffer | 190μL qH2O or TE buffer | ||
</h6> | </h6> | ||
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<b>Sequencing</b><br> | <b>Sequencing</b><br> | ||
Two main reasons: (1) after a difficult pcr/gel extraction to ensure the product is correct or (2) after cloning/biobricking to ensure no errors were introduced during PCR. | Two main reasons: (1) after a difficult pcr/gel extraction to ensure the product is correct or (2) after cloning/biobricking to ensure no errors were introduced during PCR. | ||
+ | |||
+ | <br /><br /><b> | ||
+ | Ordering Sequencing</b><br> | ||
+ | You can put in the order using the same general procedure for ordering primers. The important difference is either before or right after ordering, you need to actually prepare the DNA that will be picked up for sequencing (see Premix Specifications below). For DNA pickups for sequencing, the guy usually shows up around 2PM, so if you want an order picked up day of, be sure to have everything put together by lunchtime. This can usually be done even if you're miniprepping the sample that morning; preparing the sample for sequencing doesn't take too long unless you have a lot of samples to prepare. He always picks up the samples from room 3, there's a file box labeled "ELIM." Print out the order confirmation sheet and staple a baggy with your samples to it and put it in the box. Premix Specifications (plasmid DNA). Prepare the DNA as specified by ELIM. | ||
+ | </h6> | ||
+ | <h6 class="subindent"> | ||
+ | <br><b>For plasmids, this looks like:</b> | ||
+ | <br>500ng DNA | ||
+ | <br>0.8μl primer (one primer per sequencing reaction) | ||
+ | <br>Top up w/ qH20 to 15μL | ||
+ | </h6> | ||
+ | <h6> | ||
+ | <br> | ||
+ | For sequencing other DNA (e.g. PCR product) see the <a href="http://www.elimbio.com/Sample_Preparation.htm" target="_blank">ELIM website for specifications.</a> | ||
+ | |||
+ | <br /><br /><b>Checking the Data</b><br> | ||
+ | Usually sequencing data will be available the morning after you put in the order, sometimes early, sometimes closer to lunchtime. The results can be accessed again through the ELIM site; after signing in, there is an option to "retrieve/download sequencing data." You can either just view, or download the files. I recommend downloading all the files because you'll want to view them all anyway. Tools like ApE or Geneious will be needed to properly read the files. Each sequence read will come with a '.ab1' file that visualizes the data, and a '.seq' file that actually gives you the sequence they read. Check the ab1 file first; you're hoping for strong clear peaks, where one of four different colors represents each possible base. Typically the beginning and end of the read will look sloppy, but the middle few hundred bases should look very pretty. If the read looks pretty clean, then open the .seq file and compare the sequence to the theoretical sequence. | ||
</h6> | </h6> | ||
</div> | </div> | ||
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<div class="row"> | <div class="row"> | ||
- | <div id="precontact" class="small-3 small-centered columns | + | <div id="precontact" class="small-3 small-centered columns cells5"> |
</div> | </div> | ||
</div> | </div> |
Latest revision as of 04:23, 16 October 2014