Hello, everyone,
I posted a question about single worm PCR two months ago and got a lot of helpful replys, here I want to say thanks to the people who gave me suggestions, I very appreciate it.
I tried to change all the reagents and Taq polymerase, I even re -order the primers, It seems it is still not working. The only thing I can do now is to switch primers, I hope i can solve this problem with the newly designed primers.
Can anybody give me some suggestions which program is better for primer design? Thanks!
I just found a paper discussing the genotyping crash problem, the explanations sound reasonable. They think that if a primer which used to work very well no longer work and the PCR gradually becomes worse and worse, the very likely reason is because of the gradual built up of amplifiable DNA contamination specific to the PCR primers used. The best way to avoid this phenomenon is to separate Pre-PCR and Post -PCR.
Han DD et al., (2006) Cause and solutions to the polymerase chain reaction smear problem in genotyping.
Analytical chemistry 353(2): 296-8