Primer breakdown or template alteration

[Anonymous]

We run PCR’s for the genotyping of knockout mice. The protocol calls for three primers and Platinium TAQ. The difficulty is that over time the primers cease working. A new commerical lot of the same primer doesn’t work either. Hence the primers have to be redesigned altering them slightly. Then they too work for awhile (perhaps a month or two) and then the reactions get weaker and weaker and fade away also.

What’s happening, and, more importantly, how can we fix it?

[Daniel]

Hi Les

This sound like PCR poisoning. What occurs is you create primer dimer products which end up contaminating your lab and pipettors. So instead of amplifying the correct product the primers all end up going into making more primer-dimers. This is a problem that gets worse overtime, but as soon as you change to new primers it goes away, until the new primers become poisoned of course.

The solution is to make sure that you set-up the PCR reactions in a totally different location with different pipettors to where you run the PCR products. The idea is to get any downstream applications away from the PCR setup. One thing I have seen is people will go to a lot of trouble to run their PCR products on a gel in another room, etc., but then use their PCR set up location and pipettes to do a sequencing reaction on the PCR product! The rule is once a reaction comes out of the PCR machine then it should never come back into your set up area.

Daniel Tillett
Nucleics Support

[Author]

Posts