PCR master mix containing primers and Taq (View forum version)



Sakumi

Posted 29 May 2015 - 12:32 PM

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


Mad Researcher

Posted 29 May 2015 - 01:08 PM

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.


phage434

Posted 31 May 2015 - 12:45 PM

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.


hobglobin

Posted 01 June 2015 - 07:28 AM

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.


Sakumi

Posted 01 June 2015 - 01:01 PM

Well, thanks to everyone's reply.

 

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