problems with mix of MSP's primers - (Sep/16/2005 )
Hello all! I try to make my MSP almost for year and have a lot of problems with it. Your topics helped me so much. But now I have a new problem and dont know what to do.
Recently, I decided to chek my primers for salts wich can be prevent my MSP. I pipetted my MET & UNMET primers to the test system (with other DNA & working primers) one by one and mixes MET with MET and UNMET with UNMET.(Total I took 6 samples and 2 controls). After phoresis I saw that all the test-system with a single primer are work, but two test systems with mix of primers didn't give me any result. Is it may be that single primer doesn't inhibit the reaction? but mix of them do? Or m.b. its depend on dimers & pins? (I didn't try hot-start!)
Thank so-so for any ideas!
hot start affects specifity, but the reaction should still have worked if it were the taq
check your primers for dimer and hairpin formation
how different are the tm's of your primers?
Hi,aimikins! Thank you for your replay. My met-primers' Tm are 55 and 58 C, unmet - 54 & 58 C. I check them on OLIGO 6 for hairpins - there are no. I don't know what to think about...