Difference between revisions of "Team:Freiburg/Protocols/Colony PCR"

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<h1 class="sectionedit1"><a name="colony_pcr" id="colony_pcr">Colony PCR</a></h1>
 
<h1 class="sectionedit1"><a name="colony_pcr" id="colony_pcr">Colony PCR</a></h1>
 
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<h1> Standard protocoll colony pcr </h1>
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<h2> Standard protocoll colony pcr </h2>
 
<p> Colony PCR was tested by me (Stefan) for the insertion of an insert into pSB1C3 backbone. It is very importent to use primers with high binding affinities (such proposed by GATC) and with equal melting temperatures. <p>
 
<p> Colony PCR was tested by me (Stefan) for the insertion of an insert into pSB1C3 backbone. It is very importent to use primers with high binding affinities (such proposed by GATC) and with equal melting temperatures. <p>
 
<h3> PCR - Mastermix </h3>
 
<h3> PCR - Mastermix </h3>
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Latest revision as of 07:10, 20 November 2015

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Colony PCR

Standard protocoll colony pcr

Colony PCR was tested by me (Stefan) for the insertion of an insert into pSB1C3 backbone. It is very importent to use primers with high binding affinities (such proposed by GATC) and with equal melting temperatures.

PCR - Mastermix

µl type
2.5 Taq Buffer
1 Primer1
1 Primer2
2.5 dNTPs
0.125 Taq Polymerase
Add to 25 H2O
  • First step: 10 min 95 °C (Bacteria boiling)
  • Then follow normal PCR protocoll but mind to use a extention temperature of 68 °C (1 min/kb).

Proceeding

Aliquote the prepared mastermix to the desired numbre of pcr tubes. Pick colonies with a pipet tip and bring them into the pcr tube by scratshing on the innter tube wall and mixing. Thereafter stap with the same pipet tip into a agar plate to recultivate the bycteria used in this colony pcr. (dont forget to mark the colonies on the new agar plate). Run the PCR and seperate bands by gel electrophoresis. Have Fun.