RIPA buffer for PCR

CramCycle

Using a RIPA buffer as I will be doing westerns and ELISAs but may want to do PCR with some of the samples.

Wondering are there any interfering products in RIPA that would inhibit a PCR? or will it be fine, maybe dilute it a good bit before running.

josey_whale

I know little about using RIPA. I know a lot about PCR though. Can you tell me what's in the buffer? If it's the same as what's described on wikipedia, there's a good chance the PCR will be inhibited. The detergents in there are known PCR inhibitors. The question is - how much inhibition. Are you doing quantitative PCR?

You could test the level of inhibition by spiking a blank extract with purified DNA - I would try this first. I assume you're not doing an RT step? RT is very sensitive to inhibitors.

Two scenarios...
The level of inhibition is low - do nothing - PCR your target directly from the extract.
The level of inhibition may be high - dilute the sample in TE and then PCR. Do this if you have lots of DNA in your sample and you have a highly efficient PCR. Alternatively, extract/purify and concentrate the DNA from the RIPA extract and then PCR.

CramCycle

Thanks Josey, I was thinking of that. I might follow it up with a Phenol /Chloroform clean up after the fact, should get rid of any of the inhibitors.

My first thinking was the same as your second one, dilute in TE and go with it, hope the concentration of DNA is reasonably high and that I can dilute out the inhibitors.