Colony-PCR

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
• 20,9 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