How to check if reverse transcription went OK and there is cDNA - (Jun/21/2006 )
I have RNA and performed cDNA synthesis. I want to check if there is cDNA or not. I checked OD260 before and after cDNA synthesis, it was 0.4 before reverse transcription and 0.2 after reverse transcription, probably mix of nucleotides absorb more than strand of DNA .Is this correct?
spec'ing cDNA can be tricky and unreliable....there have been other threads here on that particular topic
to see if your cDNA is 'good', I would use straight PCR...but perhaps someone out there knows a better way?
to see if your cDNA is 'good', I would use straight PCR...but perhaps someone out there knows a better way?
I tried PCR, PCR didn't work this time, that is why i want to check cDNA
Did you check your RNA first? DId you have a look at it on a gel?
I did not check RNA by gel but I checked by OD260 and OD260/OD280 and it was OK
Unfortunately that doesn't tell you anything about the quality of your RNA. You need to run a gel to understand if your RNA gets degraded. I always run a gel and read the absorbance. I would advice you to run a bit of RNA to see if it is fine and then, if it is fine, you can do a reverse transcription.