PCR master mix containing primers and Taq - (May/29/2015 )

Hi All!

I made a mix containing primers, water and conventional PCR master mix (Taq, buffers, dNTP). I froze them at -20 yesterday, and will be using them by tomorrow. Is freezing Taq and primers together a big problem?

Thank you!

Sakumi

Sakumi on Fri May 29 20:32:28 2015 said:

Hi All!

 

I made a mix containing primers, water and conventional PCR master mix (Taq, buffers, dNTP). I froze them at -20 yesterday, and will be using them by tomorrow. Is freezing Taq and primers together a big problem?

 

Thank you!

 

Sakumi

You should prepare a master mix without adding the Taq. Taq should be added at the end. You could try using it but if the PCR doesn't work you cannot figure out what went wrong. Also, freeze-thawing your prepared master mix may result in % loss of the Taq activity as you have added the primers.

So, better add Taq when you are ready to do the PCR.

Freezing enzymes (or proteins in general) is a bad idea. This is why most enzymes are stored in buffers containing high concentrations of glycerol, which does not freeze at -20. You could add primers (in low volume) but I would avoid adding the water if you plan on storing your PCR master mix at -20.

Commercial master mixes (such as Qiagen's Multiplex kit) which already have Taq included, are also frozen and they work well. Perhaps they add a little extra Taq to avoid problems. Therefore I guess there will be no big problem if you avoid the aforementioned multiple freeze-thawing.

Well, thanks to everyone's reply.

I tried it, and the bands came up fuzzy, so I suppose I won't do that again....