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Mixing two cDNA samples into one for realtime PCR

real-time pcr rt-pcr cdna cdna synthesis

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#1 detriar

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Posted 25 May 2013 - 04:41 AM

Hello all,

I want to run my first real-time pcr assay, but first I need your help on the cdna synthesis. I'm using first-strand cdna synthesis kit from Roche, which resulting 20 ul cDNA yield per one tube. But since I think it's very 'small' result, I want to do it two times so it will resulting two tubes (20ul + 20ul) per one sample, and in the end I want to 'mix' those 2 tubes into one so I get 40 ul cDNA per one sample. Is it okay for me to do that? Or anyone can suggest another way? Because I've read somewhere that RNA from one sample must undergo cDNA synthesis at the same condition and preferably at the same time.
Thank you for your response :)

Cheers,
detriar

#2 pcrman

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Posted 25 May 2013 - 11:01 AM

Yes you can as long as the starting RNA is the same.

There are two ways of getting large amount of cDNA: one is scaling up RT reaction, the other is run several RT reactions and then combine the resulted cDNA.

#3 detriar

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Posted 26 May 2013 - 09:25 PM

Thanks pcrman
I think I'll choose the latter, since the kit doesn't recommend to scaling up the reaction, maybe it will affect the efficiency.
Cheers :)

#4 pcrman

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Posted 26 May 2013 - 10:01 PM

I agree with you. I also do several RT reactions if I need a lot of cDNA.





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