Team:British Columbia/Notebook/Protocols/PCR

From 2014.igem.org

(Difference between revisions)
m
 
(11 intermediate revisions not shown)
Line 4: Line 4:
<div class="container">
<div class="container">
<h1>Colony PCR</h1>
<h1>Colony PCR</h1>
-
<br>
 
<br>
<br>
<h2>Supplies needed:</h2>
<h2>Supplies needed:</h2>
Line 31: Line 30:
<li>Make master mix of PCR components except the DNA. Keep on ice.</li>
<li>Make master mix of PCR components except the DNA. Keep on ice.</li>
-
<img src="https://static.igem.org/mediawiki/2014/6/6e/Mount_polley.jpg">
 
-
<img scr "https://static.igem.org/mediawiki/2013/b/ba/PCR_A.JPG">
 
-
N=number of samples
+
 
 +
<img src="https://static.igem.org/mediawiki/2013/b/ba/PCR_A.JPG">
 +
<br> </br>
 +
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)
 +
<br>
 +
<br>
<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*
 +
<br>
 +
<br>
<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*
-
 
+
<br>
-
 
+
<br>  
<br>  
<li>Run PCR:</li>
<li>Run PCR:</li>
-
<ol>
+
<ol type="a">
<li>Turn machine on</li>
<li>Turn machine on</li>
Line 54: Line 57:
<li>Select appropriate temperatures and times</li>
<li>Select appropriate temperatures and times</li>
-
<ol>
+
 
 +
<ol type="i">
<li>95°C for 10s </li>
<li>95°C for 10s </li>
Line 68: Line 72:
<li>4°C forever</li>
<li>4°C forever</li>
-
</ol>
+
</ol type="i">
 +
<br>
<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>
 +
</ol type="a">
</ol>
</ol>
-
</ol>
+
<br>
-
 
+
<br>
</div>
</div>
</html>
</html>

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.


Retrieved from "http://2014.igem.org/Team:British_Columbia/Notebook/Protocols/PCR"