Difference between revisions of "Team:Penn/Notebook"

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<p><br /><strong>Protocol:&nbsp;</strong><strong>Template Preparation</strong><strong><strong>&nbsp;</strong></strong></p>
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<p><br><strong>Protocol:&nbsp;</strong><strong>Template Preparation</strong><strong><strong>&nbsp;</strong></strong></p>
 
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<li><span style="font-weight: 400;">First, one must have a </span><em><span style="font-weight: 400;">monoclonal</span></em><span style="font-weight: 400;"> E Coli colony source. This may be one of the following three things:</span>
 
<li><span style="font-weight: 400;">First, one must have a </span><em><span style="font-weight: 400;">monoclonal</span></em><span style="font-weight: 400;"> E Coli colony source. This may be one of the following three things:</span>
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<li><span style="font-weight: 400;">This water/colony mixture will be the &ldquo;template&rdquo; for your PCR reaction. Next, set up your reaction mixture and run the cycler protocol. Follow this up by running a gel to examine the PCR amplicon lengths.</span></li>
 
<li><span style="font-weight: 400;">This water/colony mixture will be the &ldquo;template&rdquo; for your PCR reaction. Next, set up your reaction mixture and run the cycler protocol. Follow this up by running a gel to examine the PCR amplicon lengths.</span></li>
 
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<p><strong>Reaction Preparation</strong>&nbsp;</p>
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<p><br><strong>Reaction Preparation</strong>&nbsp;</p>
 
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<li><span style="font-weight: 400;">1 uL Template</span></li>
 
<li><span style="font-weight: 400;">1 uL Template</span></li>
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<li><span style="font-weight: 400;">7 uL H2O</span></li>
 
<li><span style="font-weight: 400;">7 uL H2O</span></li>
 
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<p><strong>Cycler Protocol-&nbsp;</strong>The cycler protocol is fixed except for the extension time. Set the extension time for 1 min per kb of desired amplicon at 68C, plus a little bit of buffer time. For example, for a 2 kb amplicon, set the extension time to 2 min 30 seconds.</p>
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<p><br><strong>Cycler Protocol-&nbsp;</strong>The cycler protocol is fixed except for the extension time. Set the extension time for 1 min per kb of desired amplicon at 68C, plus a little bit of buffer time. For example, for a 2 kb amplicon, set the extension time to 2 min 30 seconds.</p>
 
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<li><span style="font-weight: 400;">95&ordm;C for 6 minutes</span></li>
 
<li><span style="font-weight: 400;">95&ordm;C for 6 minutes</span></li>

Revision as of 02:02, 17 September 2015

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Colony PCR


Protocol: Template Preparation 

  1. First, one must have a monoclonal E Coli colony source. This may be one of the following three things:
    1. A glycerol stock made from a SINGLE colony on an agar plate
    2. An overnight culture in LB media (+ antibiotics if appropriate) started from a SINGLE colony on an agar plate
    3. A SINGLE colony on an agar plate
  2. Label a sterile eppendorf with the strain number and assign a unique colony number for each colony picked. Pipet 20 uL of sterile water into the tube. If the e coli is coming from sources (1) or (3), use a clean, sterile pipet tip to scrape a tiny bit of bacteria (even the very smallest amount is okay) and then pipet up and down into the waiting 20 uL. If the e coli is coming from source (2), pipet 1 uL of the LB culture into the water. Mix well.
  3. This water/colony mixture will be the “template” for your PCR reaction. Next, set up your reaction mixture and run the cycler protocol. Follow this up by running a gel to examine the PCR amplicon lengths.


Reaction Preparation 

  1. 1 uL Template
  2. 10 uL 2x Taq Master Mix (Long term storage in freezer, will last one month at 4C)
  3. 1 uL FWD Primer (@ 10 uM)
  4. 1 uL REV Primer (@ 10 uM)
  5. 7 uL H2O


Cycler Protocol- The cycler protocol is fixed except for the extension time. Set the extension time for 1 min per kb of desired amplicon at 68C, plus a little bit of buffer time. For example, for a 2 kb amplicon, set the extension time to 2 min 30 seconds.

  1. 95ºC for 6 minutes
  2. 30x [95ºC for 30 sec, 55ºC for 30 sec, 68ºC for 1 min/kb amplicon]
  3. 68ºC for 20:00 min
  4. Hold at 4ºC