I added hot star master mix and primers with my samples to be ready for PCR and keep it in deep freezer, will this affect the result??
my dear , the frozen and thawing the master mix may be destroyed the taq polymerase , therefor you should ran your sample immediately.
theoretically not because of the polymerase.. but I have done that and it actually worked!
best to keep the mixture in a 4C fridge. It should be fine and avoids the problems of freezing the enzyme. I have stored pcr mixes for 72 hours at 4c and run pcr and it has worked many times. It used to be common practice to do this at Genethon when they were running mapping sets for very many researchers about 30 years ago
In my previous lab, we used to buy the master mix (containing everything) and store in freezer! When we need to run a PCR, we just add the template to it! You can go ahead and try it! If it works for you, you can continue to do so in the future!
Dear Soha, the hot start master mix is already designed to store below -20 C, this freezing temperature and even the room temperature when you need to thaw this master mix for your PCR, the enzyme remains stable as it will only activate at certain temperature during the PCR run. That is why, we use hot start MM so acquire the maximum polymerase activity in PCR without any loss. So temperature variations or freeze thawing will not have any effect on hot start master mix. You can prepare it as per your need in aliquots and mix with your primers and store at 4 C mainly but even storing below 0 C it should work fine. The only thing your template (DNA or cDNA) may be affected by multiple freeze thaw cycles, so add your template or sample only at the time of run & avoid its storage with your master mix preparation at lower temperatures.
I've done it and it works. Make sure you seal your plates really well.
Actually, I do that with my master mix and there is no problem. I based on the ability of the enzyme to be stable at heat cycles with PCR also that its activation point is above 90C, but I store the mix on storing temperature of the master mix on 20C .
How about on MyTaq One Step RT PCR kit? Does it work the same? My sample is RNA sample and have lots of sample to be run. Can I store overnight after I add rna sample in my master mix with primer? Then, the next day I run in pcr machine.
thank you for helping me out..
It will be best if you keep the preparation in the fridge (chill at 4 degrees). But add your template time you want run the PCR.
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