PCR - No bands anymore in well functioning PCR - (Dec/21/2004 )

In our lab we do a lot of genotyping PCR of transgenic mice. Since 2 months two of our PCRs don´t work anymore. No bands at all (sometimes even the positive control is very weak or disappeares) , I really checked everything I could think of: new primers, same primers from different company, new dNTP, new dilution of dNTP, new Taq, HPLC-water new batch, DNA-isolation with different methods, OD for DNA concentration. The un-genotyped mice are piling up and my problem grows. Does have anybody any more suggestions!

Thanks in advance
Lea

use another PCR machine!

blood sacrifice.

or play with the Mg concentration... or alter the temps just a little (gradient machine)... try using a fresh batch of buffers and enzyme... let someone else make it up... put in more DNA.... put in less DNA...

We already used 2 other PCR machines and a gradient cycler and all new batches of buffers, dNTPs and a new batch from the old Taq as well as a new Taq supplier and even an PCR enhancer.
Maybe playing with DNA amount can help.

Thanks a lot for the recommendations.

Lea

Did you change the method of DNA extraction?

hey

do you check whether heating of buffer in electrophoresis, if so change the buffers regularly and try one more time.
Is your EtBr is working properly, are exposed much time to light

regards

hi,we had met the same problem in our lsb too!we sloved by using PCR additives(BSA,DMSO,Tritonx-100,etc),why not have a try?