How long can I store at - 30°C my PCR mix? - (Jun/16/2014 )
Hi guys, I´m new in the forum. Hope you can help me. We´re doing an assay of RT-PCR to get the expression of miRNAs 376c and 141. Today I did a PCR mix (Buffer 5X, MgCl2, dNTPs, Oligos, Taq polimerase and free nuclease free H2O) but I don´t have enough time to run my PCR. How long can I store at - 30°C my PCR mix?
Probably years. PCR master mixes are essentially this.
Are you sure that you adenylate your miRNA to be reverse transcrped?
I think at 4 C for more than a week
Thanks!! The PCR has all the ingredients to run the PCR except my sample. So, I suppose that I can it use for a long time right?
Mohamed: I didn´t adenylate my miRNAs, what´s the importance to do it??
Which Oligos that you use ? If you are using Oligo dt then u need to adenylate ur product
I´m using specific oligos to run rt and pcr. Is necessary adenylate my mirnas? I guess is only necessary for random primers right?
I´m sorry if I ask very basic questions, but it´s the first time I run pcr. Thanls!
If you are using a poly-T primer (sometimes called an oligo dT primer) for your RT reaction, it will only bind to RNA fragments containing a 3' poly A tail. Most eukaryotic transcripts will have this, but your miRNA fragments may not. That is the only issue which was pointed out.