Does anyone prepare big master mix batches (that means primers, buffer, magnesium chloride, dNTPs, water; so everything except for the Taq) to final concentration for genotyping purposes?
What I mean is, what happens if I prepare 1000 reactions, aliquot it and then unfreeze the reactions I need to run the genotyping PCR, add taq, and then use? Do the dNTPs or the primers get ruined if they are frozen at low concentrations?
Hi,
You can make up a master mix like you suggest but you should aliquot the mix to the volume of your normal PCR run. dNTPs will be ruined by freeze thaw cycles. There are manufacturers who sell master mixes with all the reagents added except the enzymes and they work fine. I prefer, however, to make up my mixes fresh on the day.
Hi,
I agree with Roy that you should aliquot your mix, but I would even put the taq in the it.
During my PhD, I worked in a pathology department and we used to aliquot PCR mix (with everything in it) in 0.2ml tube-strip (for STR profiling - meaning multiplex PCR) for day to day routine experiments.
It worked perfectly fine. The only thing was avoiding freezing thawing cycles as suggested, and making sure that the reagents were actually working (not using old reagents that you don't know if they works... ;-) )
Best,
I also have good experience with making big batch of master mix and aliquot it. It worked well for me (tested after several weeks also), but in my case I avoided adding the Taq, personally.
Thank you everyone!
Yes I was thinking of leaving the Taq out, since it's what could suffer the most (beside the dNTPs)
Thank you everyone for the answers!
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