Colony-PCR

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Current revision (02:12, 30 August 2013) (view source)
 
Line 7: Line 7:
*0,2 µl Primer rev
*0,2 µl Primer rev
*0,04 µl Taq Polymerase
*0,04 µl Taq Polymerase
-
*20,9 µl Water
+
*8,36 µl Water
==PCR Program==
==PCR Program==

Current revision

Instead of plasmid or genomic DNA, you can use a colony from the agar plate as template DNA. You should use a sterile toothpick, pick into the colony and spread it in the PCR tube.

For a 10 µl reaction (Taq Polymerase Peqlab):

  • 1 µl Buffer S
  • 0,2 µl dNTP's (10 mM)
  • 0,2 µl Primer fw
  • 0,2 µl Primer rev
  • 0,04 µl Taq Polymerase
  • 8,36 µl Water

PCR Program

  • 95°C 5 min

35 times the following cycle

    • 95°C 1 min
    • 55°C 30 sek. (°C depends on your primers)
    • 72°C 1 min/kb

cycle end

  • 72°C 10 min
  • 10°C hold
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