Set up 1x PCR buffer mix

5X Green or Colorless GoTaq® Flexi Buffer: 10µl (1x)

PCR Nucleotide Mix, 10mM each: 1µl (0.2mM each dNTP)

upstream primer: 2µl (1.0µM)

downstream primer: 2µl (1.0µM)

GoTaq® G2 Flexi DNA Polymerase (5u/µl): 0.25µl (1.25u)

MgCl2: 2ul (1 mM)

Nuclease-Free Water to: 50µl

Note: Can reduce the final volume to 25µl to save reagents.

Using a sterile toothpick or 200µl pipette tips, gently touch a single bacterial colony and dip it into the PCR buffer mix.

PCR reaction

Initial denaturation: 95°C, 2 mins

*Denaturation: 95°C, 1 min

*Annealing: 55°C, 30 sec

*Extension: 73°C, 1 min for every 1kb of amplification

Final extension: 73°C, 5 mins

Storage: 12°C or room temperature depending on when the products will be analysed

*20 to 30 cycles (Generally 20 cycles is more than enough to amplify)

Note: Increasing the annealing temperature will increase the specificity of the PCR reaction.

Important: Run gel electrophoresis immediately as PCR products will normally be degraded due to the presence of DNAse from the lysed E. coli. PCR products not recommended for long term storage.