Primer Trouble Shooting - (Mar/07/2010 )
Hello every1... i need help regarding primer trouble shooting......
I am working in a project of HBV genotyping. I am following a protocol from a published paper for type specific genotying ( it involves nested PCR). Well few mothns before (about three months before) things were working fine that suddenly my PCR stoped working. I trouble shooted every thing by using new set of reagents and stuff except the primes, but to no avail so at last ordered a new set of primers. The new set of primers worked fine as i had otpimized the conditions and TM by using gradient PCR, however after a week of the arrival of new primers....again the same thing started happening.. i am not geting any amplification in the first round. ... no bands... can any1 help me out....its driving me cazy
How are you dissolving your primers? I recommend TE rather than water. Your water may be acidic, degrading your primer stock. The amount of EDTA added with reasonable concentration primer stock is minimal and well worth the preservation of your primers (and templates).
well i had dissolved my primers in water (Qaigen nuclease free water). So there aint any doubt regarding the pH issue......
Yes, there is doubt. The pH of pure water is very poorly controlled (no buffering). CO2 absorbed from the atmosphere can easily tip it to the acidic side. There is a reason for buffers! You can't measure the pH either, since the measurement process introduces more impurities than are found in the water to start with, dramatically altering the pH.
Definitely dissolve them in TE - also how are you storing them? We generally aliquot out a small amount so that the whole thing is not constantly being frozen and refrozen.
well 10nx 'phage434' 4 ur help and advice...as you guys are asking me to use TE... so will be using TE in future when dissolving primers .... and the funny thing is that they have statred working again...heheheheh dont knw what was the freaky thing abt it..but they have started working ...
i do practice aliquoting them out a bit in order to pevent continous thawing....dont knw hw have they have started working again