4 replies to this topic

[Kaylo]

Hi all,
I am planning some TaqMan real-time PCR to detect microRNA in very precious, limited samples. I ordered the 2x Universal Mastermix from Applied Biosystems.
This arrived to our institute and someone else from the lab collected it from the delivery dock for me - and then put it in the freezer rather than fridge...
Does anybody have any tips or experience about how sensitive this reagent is to freezing and thawing before use? Will the enzyme activity survive okay, or is this likely to have killed the enzyme or the dye?
I am nervous not only because of the cost of the reagent itself but also the time and more especially the samples (we have only small amounts of starting material, which have taken months to collect).
I'm hoping someone else has had this problem before!
Thanks in advance for any help.

[leelee]

I don't know about that particular product, but I suspect that the single freeze thaw will probably be ok. I store my Roche qPCR master mix at -20 then thaw one bottle at a time (keeping it at 4C after thawing), and I've never had problems.

Do you have a positive control you can test?

[leelee]

You could also try emailing Applied Biosystems and see what they say? That would probably be my first step.

[Kaylo]

Yes, I've spoken to the local rep (who helpfully saysthat the instructions say store at 2 - 8degreesC, so it shouldn't have been frozen) but might try their tech specialists.
The plan had been for a 384 well plate experiment, but agreed it would be safer to try a few first in parallel with same samples run with the old mastermix, before plunging in to the whole set.
I was hoping someone would tell me that they'd made the same mistake but it still worked so I'd be fine! This whole series of experiments has been a technical disaster from start to finish, so I'm nervous!

[Kaylo]

Okay, just spoke to the tech specialist from AB. He thinks it should be fine - one round of freeze/thaw might make a small percentage effect on the efficiency, but should still work. Obviously multiple freeze/thaw rounds would not be recommended. His tip was that if it thawed and looked fine then it should be good to use, but if I could see any crystalline precipitation then that would be a worry.
Yippee, no precipitate, so still good!
Experiment had better work now