Unpredictablity of PCR product - (Mar/14/2011 )

Hi. I was wondering why sometimes I seem to get PCR product using the same protocol and other times nothing shows up. The DNA that I use is a linear plasmid DNA cut with MssI and has been in storage for 2-3 years. I was wondering if there is any other factor other than degradation? I vortex and spin down the DNA. I am not sure what is wrong. Thanks.

Any help would be appreciated.

Hey, vortexing is not a good idea, because it breaks DNA, centrifugation shouldn't be a problem...

SOS response on Mon Mar 14 21:44:52 2011 said:

Thank you. I was wondering if Plasmid DNA could also be affected. The reason I asked was the DNA I am using is 2-3 years old. Should I be extra careful and just flick it before adding to my PCR tube? Thank you.

A common problem is using too much DNA in your PCR reaction. Try reducing the amounts used 10x or 100x.

I doubt the vortexing is a problem, I vortex my DNA all the time and I've never had an issue.

phage434 on Tue Mar 15 02:26:20 2011 said:

Hi Phage,

I was wondering how much is "too much" DNA and what are the effects of having too much? Thnaks!