cDNA - (Jun/10/2005 )

The protocol:

25 ml SYBR Green Mix (2x)
0.5 ml liver cDNA
2 ml primer pair mix (5 pmol/ml each primer)
22.5 ml H2O



“0.5 ml liver cDNA”; Do I have to treat the Reverse transcriped reaction to get rid of RNA and buffer’s stuffs before using the cDNA for PCR? When it says “0.5 ml liver cDNA” does it mean that from the RT reaction I take out 0.5ml RT reaction or should I also calculate the concentration of cDNA and then based on it take out 0.5ml cDNA and not 0.5mll RT reaction?


Thanks again!

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hi
assuming that one of your primers can hybridize with RNA, i would recommend you to get rid of it.

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Dude, mL or uL?

Other wise that is a massive cDNA rxn.

I am going to assume uL.

Here is what you do. Dont purify the cDNA rxn. Take some liver RNA, run the reverse transcription rxn in the absence of Reverse Transcriptase and then put that thru the rt-pcr rxn same as the samples that did get the reverse transcriptase. Look at that amplification curve carefully. If things are going corrrect there should be at least 7-10 cycle difference between the one that did not get the RT vs. the ones that did..

Call that background. Since real time is based on a doubling of product every cycle, actual product is that Ct value minus background (sample without rt) and since it is a square funtion, background should be minimal.

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Anyone who can answer this question? Besides should i sequence the cDNA before running in PCR?



Thanks again!

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Thanks fred!

Do you know how i can get rid of this RNA as well as most other buffer components to get a pure cDNA solution?

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