Running multiple PCRs in a day - (May/06/2010 )
Having gone through days of many PCRs a day, I realised the damage of freeze thawing primers and dNTPS! I would recommend to anyone doing multilple PCRs a day, to use a fresh (or not recently thawed) aliquot of primer and dNTP for each run. WHen you make up dNTPs and primers, aliquot them to several tubes (and each time you use a tube, mark it so you know how many times you have thawed them).This way, you dont use one aliquot over several days, and rotate between aliquots. For some reason if I used the same tube consecutively for a few days, sometimes it worked and sometimes it didnt leaving me puzzled with a blank gel! Just thought it might useful for beginners at PCR!
If you run more PCRs in a day then you should let the primers and stuff on ice and just don't freeze it again. Freeze-thaw cycles make more damage than leaving it on ice for some time.
I leave my primers in the fridge when I'm using them every day.
I have tried leaving it on ice or fridge the whole day and freezing them at the end of the day. I havent tried leaving them in the fridge for a few days because I wasnt sure how the dNTPS would handle it
This may sound crazy but, besides Taq, I put all my working stocks required for PCR (primers dNTPs MGCL2 10x buffer) in a 4C fridge. I had been doing this for months and I have had no problems with my PCR.
That s great for you! For me, it varies between different primer sets... some seem to be more sensitive than the others wrt freeze/thaw... so i uniformly store them at -20
For me, i do a master mix and aliquote into each individual tube and frozen it. another master mix which consist of h20 and taq will only be mixed before i run my pcr, and will add together with template into the frozen tube to "melt" it.
So far, no problem for my multiplexes.
Adrian, have you created a new way to make a hotstart?
Maddie on May 12 2010, 09:35 PM said:
But one thing for sure, I'm using hotstart Taq.