colony PCR inconsistent - (Aug/30/2010 )

my colony PCR result is very inconsistent, sometimes i do get the product and sometimes i dont. I check the primer set and it is alright, i also checked all the reagents i used for the PCR they all work perfectly.

the funny thing is when i do colony PCR for testing purposes (to check for whcih colony has insert) it never fails but when i try to amplify it in bulk for sequencing this happen.

The usual problem is too many cells. Pick a colony with a 10 ul tip, and swirl into 50 ul of water (scrape the side of the tube). Spot 2 ul onto a master grid petri dish with the same tip. Use a different tip to transfer 0.5 ul of the water into a 10 ul PCR reaction with your primers. Heat at 95 C for 5 minutes before normal PCR cycling.

I agree with phage434's recommendations. I also never do PCR to check for an insert directly from colonies arise on the original transformation plate -- patch some of the transformants to a fresh plate, grow these overnight, and do your colony PCR from them, first resuspending a bit of growth in 50 ul of water, as mentioned above.

Always amplify the junction between DNA fragments. PCR is sensitive enough to amplify naked DNA sitting on the agar plate (leftovers from the ligation mix).

Only the junction between DNA fragments is unique to a ligated plasmid.

This is an alternative to what Homebrew does which is to spot the colony onto a clean plate.

perneseblue is correct, I should have written "I also never do PCR to check for an insert directly from colonies arise on the original transformation plate if my screening primers are both internal to my insert -- patch some of the transformants to a fresh plate...".