Team:British Columbia/Notebook/Protocols/PCR

From 2014.igem.org

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<h1>Colony PCR</h1>
<h1>Colony PCR</h1>
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<h2>Supplies needed:</h2>
<h2>Supplies needed:</h2>
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<h2>Colonies</h2>
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N=number of samples
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N=number of samples<br>
*Add about extra amount worth two samples to account for water control and pipetting error.
*Add about extra amount worth two samples to account for water control and pipetting error.
(if regular PCR is done use ca.50-200ng template DNA and use Phusion and 10x Phusion buffer instead of Taq)
(if regular PCR is done use ca.50-200ng template DNA and use Phusion and 10x Phusion buffer instead of Taq)
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<li> Aliquot 25µL per PCR tube, keep tubes on ice.</li>
<li> Aliquot 25µL per PCR tube, keep tubes on ice.</li>
*Following steps must be done near the flame*
*Following steps must be done near the flame*
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<li>Touch toothpick/ pipet tip/ loop tp colony, then index plates, then dip in the PCR tube.</li>
<li>Touch toothpick/ pipet tip/ loop tp colony, then index plates, then dip in the PCR tube.</li>
*Turn of flame*
*Turn of flame*
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<li>Run PCR:</li>
<li>Run PCR:</li>
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<li>Turn machine on</li>
<li>Turn machine on</li>
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<li>Select appropriate temperatures and times</li>
<li>Select appropriate temperatures and times</li>
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<li>95°C for 10s </li>
<li>95°C for 10s </li>
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<li>4°C forever</li>
<li>4°C forever</li>
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<li>Once finished, remove the PCR tubes from the machine.</li>
<li>Once finished, remove the PCR tubes from the machine.</li>
<li> Verify PCR products agerose gel or store PCR tubes at 4°C or -20°C.</li>
<li> Verify PCR products agerose gel or store PCR tubes at 4°C or -20°C.</li>
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Latest revision as of 23:39, 17 October 2014

2014 UBC iGEM

Colony PCR


Supplies needed:


  • PCR tubes
  • BioBrick PCR primers (VF2, VR)
  • Taq polymerase
  • 10x Reaction Buffer
  • 10mM dNTPs
  • dH2O

Steps:

  1. Make master mix of PCR components except the DNA. Keep on ice.


    2. N=number of samples
    *Add about extra amount worth two samples to account for water control and pipetting error. (if regular PCR is done use ca.50-200ng template DNA and use Phusion and 10x Phusion buffer instead of Taq)

  2. Aliquot 25µL per PCR tube, keep tubes on ice.
    3. *Following steps must be done near the flame*

  3. Touch toothpick/ pipet tip/ loop tp colony, then index plates, then dip in the PCR tube.
    4. *Turn of flame*

  4. Run PCR:
    1. Turn machine on
    2. Load samples in machine
    3. Select appropriate temperatures and times
      1. 95°C for 10s
      2. 94°C for 30s
      3. 60°C for 30s
      4. 68°C for 1min per kb of expected product (round up)
      5. Repeat 2-4 30times
      6. 68°C for 20 mins
      7. 4°C forever

    4. Once finished, remove the PCR tubes from the machine.
    5. Verify PCR products agerose gel or store PCR tubes at 4°C or -20°C.


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