Reaction Mixture Use the following table as a checklist for preparing the reaction in reaction tube (1):
PCR supermix
VF2
VR
colony template
Colony PCR
9 µl
0.25 µl
0.25 µl
0.5 µl
PCR conditions Program a standard thermocycler to run the reaction using the following parameters: Initial denaturation
Denature: 95°C, 15 mins
Thermocycling
No. of cycles: 39
Denature: 94°C, 30 secs
Anneal: 56°C, 30 secs
Elongate: 68°C, 60 secs
Elongation time : 1 min per kb of expected product. I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time. Termination
Elongate: 60°C, 20 mins
Hold: 4°C, until removed from machine
Perform agarose gel electrophoresis of appropriate quantity of PCR products mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 55 mins
