Problem in Patch PCR for Site-Directed Mutagenesis - (Aug/28/2006 )
Hi,
I have been doing Patch PCR to get a site-directed mutagenesis. The mutant base lies in the overlapping primers. The first two PCRs using the two end primers and the overlapping primers work very well. I got the desired bands. Then I mixed the two purified PCR products and made 1:100 dilution as template to do the third PCR using the two end primers. I got nothing from the third PCR. Then I doubted maybe the overlapping primers are not good and ordered two new overlapping primer. But still got the same result. 
Is there somebody having experience in doing Patch PCR? Could you give me some help? Thanks in advance.
Dream
Hmm... there is not enough info here to make a diagnosis.
When you mean nothing, do you mean a blank gel? Or a smear with nothing in the right size. How big is the fused product? The overlap size and melting temperature.
But at the risk of looking like an idiot, i would say that you have a problem of fusing the two products... ie the PCR conditions are not right. If the annealing temperature is correct and the correct polymerase for the product lenght is being used, I would suggest that you lower the extention temperature, try 70 Celsius. Make up for lower extention temperature by increasing the extention time.
Well hope that works. If not drop more info and maybe an answer will come.
When you mean nothing, do you mean a blank gel? Or a smear with nothing in the right size. How big is the fused product? The overlap size and melting temperature.
But at the risk of looking like an idiot, i would say that you have a problem of fusing the two products... ie the PCR conditions are not right. If the annealing temperature is correct and the correct polymerase for the product lenght is being used, I would suggest that you lower the extention temperature, try 70 Celsius. Make up for lower extention temperature by increasing the extention time.
Well hope that works. If not drop more info and maybe an answer will come.
Perneseblue, thank you very much.
"nothing" means nothing in the right size, having two samll unspecific bands. The fused product should be 2kb. The overlap size is 23bp and the Tm for the overlapping primer is about 58 degree.
Now we are trying to use the site-mutagenesis kit to do this. I hope this will work. Thanks.
Have you tried lowering the annealing temperature during your second round? A trick which has worked for me in the past is to mix up the second round in two separate tubes, with each primer in one of the tubes. Run a few cycles of this, which produces linear amounts of ssDNA for each of the halves. Mix the solutions and complete the PCR process.
But I'm guessing either that your primers are binding to multiple locations or that your annealing temperature is too high. Try 55C, it almost always works. Run the extension at 68, and make sure it is long enough (2 minutes?).
But I'm guessing either that your primers are binding to multiple locations or that your annealing temperature is too high. Try 55C, it almost always works. Run the extension at 68, and make sure it is long enough (2 minutes?).
Thank phage434. I like your trick. It's new to me.
I changed to use the site-mutagenesis kit. It should be much easier.
Thanks again.
That is one cool trick phage454! 
May I ask how many cycles do you run to make the ssDNA?
Thanks for sharing this idea.