Sometimes i get bands, sometimes i get nothing for my PCR - Strange PCR Results (May/14/2005 )
I am new at PCR (standard) and have been doing it for 3 weeks for genotyping purposes. I prepare the master mix in the order of adding H20, DMSO, PCR Buffer with MgCl, 2 primers. Then I load my gene samples (1-1.5 ul). Jus before I add my master mix, i add dNTPs and taq polymerase, mix well, then add 29 ul to each sample. I am using PCR plates and my cycler has heated lids. The number of cycles vary depending on the gene, usually around 33 cycles. The program has been optimised and it always works for my colleagues but not for me.
After running gel electrophoresis, there are 2 kinds of results i get. Either there are bands or no bands. Sometimes for the same PCR run, some samples show bands and some none at all. My colleague did PCR for the same samples and it turns out that I did not get some bands that I am supposed to for some samples. Sometimes I am not supposed to get bands but i get it. In short, my PCR results are inconsistent and I don't know how i can make it work consistently. I have no idea what is wrong too since I am now following exactly how my colleagues do it. I do everything in the cold box and ice block.
Any idea what could be wrong and how i can improve my PCR?
A while ago, someone raised the same problem. It may help to read the discussion of that thread at http://www.protocol-online.org/archive/posts/4173.html