colony PCR-sometimes works, sometimes doesn't :( - (Aug/02/2006 )

hi folks,

I need to PCR check some bugs for successful shRNA insert into a vector. I'm 99% sure the bugs have a vector in as the negative control plate was virtually blank, but the colony PCR itself is very unreliable. I use freshly-plated bugs (maybe 18 hours on the plate) and have tried...

1) picking colonies & putting these directly into the PCR mastermix
2) picking colonies into 100 uL water, 95C for 5 min then 5uL into PCR

both work maybe 20% of the time & it's wasting a load of effort. Anyone got any tips for reliable & reproducible colony PCR ?

thanks !

Are these E. coli? coi works with these techniques, but other bugs can be problematic. Use very small amounts. I think 5 ul is too much. I put 1 ul of a 100ul dilution of picked colonies in a 20 ul reaction. I don't bother with any treatment, since the PCR initial denaturation (5 minutes to 15 minutes at 95) does that for E. coli. You need quite a few cycles -- I run 40 cycles. You might want to verify the robust amplification of your product to make sure you are not close to failing on the annealing temperature.
Make sure it works for a few degrees each way from the annealing temperature you are using.

You want one of your primers to be in the insert and one in the vector to avoid false positives.

Triton 0.1 % helps lyse the cells, I find, so if your buffer lacks it or another detergent (like Tween, NP, etc.), then add it.

I run 35+ cycles.

Good luck!

-Matt

I do more or less the same than phage434, I pick a colony (not the entire colony, just touch it to bring just a little amount of bacteria with a tooth ) in 50 ul of H2O and then, I use 1 ul in a 10-ul PCR reaction (for saving taq). I run 35 or 40 cycles. It always always works for me.