How to know there is cDNA? - (May/01/2007 )

I isolated RNA using TRIzol. The ratio of 260/280 is 1.9. Agarose gel did not show obvious degradation. I use DNase to get rid of DNA and then did cDNA synthesis using first-strand transcription. I also use RNase H to clean my cDNA. But the rt-PCR did not go well. I cannot get single clear dissociation curve. Now I want to figure out whether the problem is due to the RT step or the PCR step. so, How to check if reverse transcription went OK and there is cDNA? THanks!!

Use controls

1. Do a control PCR using your genomic DNA so that you know that your PCR is working.

2. Do a control RTPCR using a housekeeping gene.

If both these work then you know that there is cDNA that can be PCR amplified.

Cheers

Daniel

DNA sequencing troubleshooting help

Thanks!
So there is no way to identify cDNA directly?

Sure, there is. Think over.
E - exon
I-intron
> and < - primers


--EEEEEEEE-IIIIIIII-EEEEEEEEE-- DNA
. . . . . . . > . . . . . . < . . . . . . . primers
no product, the range is big




--EEEEEEEE--EEEEEEEE-- cDNA
. . . . . . . .> . <. . . . . . . primers
there is product

GOOD LUCK:)

Sometimes....some DNA "survive" to the RNA isolation.....
You can make 2 RT-PCR reaction per sample....the same steps for both....the only difference is one of them does not have the retrotranscriptase in it. Then you run PCR for both.
If you have the same bands in both, it is probably genomic DNA what you amplified.....if you have 2 bands in the sample where you used transcriptase and only one in the sample witout it...then the lighter band is cDNA and the other is genomic DNA.
Hope it helps

i am with solmaniar. i like to use this control most. another one is you run PCR using specific primer designed to amplify you cDNA