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How long can I store at - 30C my PCR mix?

pcr dna rt-pcr

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#1 qfbmendieta

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Posted 16 June 2014 - 01:49 PM

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?

Thanks!!!



#2 phage434

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Posted 16 June 2014 - 01:58 PM

Probably years. PCR master mixes are essentially this.



#3 Mohamed 1984

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Posted 16 June 2014 - 02:36 PM

Are you sure that you adenylate your miRNA to be reverse transcrped? 

 

I think at 4 C for more than a week 



#4 qfbmendieta

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Posted 19 June 2014 - 02:54 PM

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??



#5 Mohamed 1984

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Posted 21 June 2014 - 05:34 PM

Which Oligos that you use ? If you are using Oligo dt then u need to adenylate ur product 



#6 qfbmendieta

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Posted 24 June 2014 - 10:12 AM

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!



#7 phage434

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Posted 24 June 2014 - 10:29 AM

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.







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