I am unable to amplify the band of my interest from the new cDNA, whereas the old cDNA used to give a very prominent band at 60 degrees. I have repeated the reaction umpteen number of times, with all the new reagents but no amplification still. What could be the problem?
The first thing to think about would be if your reverse transcription has worked (assuming you did that to generate cDNA) for sure. If yes, than your PCR has a problem, if not, it can either be RNA quality or cDNA preparation.
are you using a one-step RT-PCR kit (i.e. Invitrogen Superscript III) or conducting individual reactions? Also, how are you extracting the RNA...kit or manual (chloroform/phenol)? Each of the these techniques and/or methodologies have caveats associated with them and often times where one method will work under one set of conditions, a separate isolation/amplification may require the other...
Which kit did you use?What was your starting material (RNA)?Usualy the most kits create always cDNA, if you have good quality RNA. The cDNA created from the same RNA?If yes, probably the RNA degradated.
is your RNA coming from the same starting samples?If yes, did you try to check if the RNA is degradeted? i usually run 1 or 2 ul on a gel for 30 mins, and this is enough to have an idea. if not, maybe the expression is low (or absent) in the new one and you need more cicles. try also to use your new cDNA PCR product as template for a new more selective PCR.
Also, if you have already not, ensure that the starting amount and quality of RNA for both cDNA synthesis reactions are comparable. If the transcript is of low abundance and one tube had a much lesser RNA to start with, you might just miss the transcript.
I extract RNA from the paddy and i can't get the cDNA band after amplification. What would this cause ? is it the temperature or the sample?
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