For doing PCR, instead of preparing the PCR mater mix solution each and every time, is it advisable to prepare it in a larger quantity and store it at -20C so that the PCR can be set up faster. Please enlighten me with the pros and cons of doing it this way.
Aparna Varma this should be absolutely feasible. Think about it. Companies, such as Promega, sell PCR mastermix stored in -20C.
If you avoid freeze thawing you should be fine.
hi
there is a stabilizer( such as glycerol ) and enhancer ( such as DMSO ) In commercial master mix.In my opinion, test in low volumes.
good luck
Thank you Reza Hadavi Sir. I will try it. Can you suggest me the minimum concentration to start with?
Master mix can be prepared (without enzyme) and stored as you mentioned or lower temperature.
I also do it and get result.But you should not add the enzyme in advance,there is chance of reduction in its activity, if kept in low temparature for a long time.
Thank you Mohammad Kabir Ahmed Sir and Soumyajit Biswas Sir. I will surely consider your suggestion.
I have the experience of using Ampliqon Taq DNA Polymerase Mix Red-Mgcl2 1.5 mM and have a good result for them. The use with colorful type mastermix is so comfortable.
Hello Aparna, You can do it, no problem. But from my experience, I am suggesting you that, please add the enzyme just before performing PCR. Because, there is a chance of reduction the activity of enzyme.
Hi,
we are doing this routinely. You can prepare mastermix without enzymes as suggested above. The reason is Taq polymerase (hot start or whatever generation) should be not be stored in a frozen state. If you prepare a master mix with required components and store at lower temperature certainly it will get into a frozen state... It will affect the enzyme activity. Thats why first/second generation Taq provided in separate aliquot and it is not in a frozen state (even in -20). Most importantly one time thaw is allowed to get consistent result. Prepare master mix in larger volume, aliquot it according to you requirement, thaw one time and add enzyme once thawed (gradually).
Thank you Narayanan Kalyanaraman Sir for the explanation. I will surely keep this in mind.
You can set up the entire plate (enzyme, buffer, DNA, etc.), seal it well, and put the whole thing in the freezer overnight and put it in the thermocycler the next morning.
Or you can buy a good "hot start" polymerase master mix. It only takes a few minutes to thaw. Just put it on your bench while you do your calculations, label plates, etc.. It will be thawed and ready to use when you are ready to set up your reactions.
No need to list "pros and cons". Just buy quality reagents and use them!
Hy Aparna Varma
As everyone suggesting make the Master Mix without enzyme(like Taq or Phusion) and aliquot in PCR tubes.So while setting up PCR, add your template, primers and Enzyme directly into the Mix which prevent some errors.
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