invitrogen Superscript one step RT PCR kit - Efficiency of kit (Apr/18/2005 )
I am using superscript one step rt pcr kit (invitrogen) to amplify the cDNA from chick RNA. But somehow i am not getting the amplification. the quality of RNA was good.i've checked products on 1% agarose gel . all products were of same size and concentration in conrols and experimental reaction. product was also seen in control containing no enzyme .only the product was not seen in control reaction containing primers and enzyme. I think that problem might be in cDNA synthesis. and what i'd seen on gel was just RNA.
So, please if u have any suggestions, i will be highly obliged to receive.
What do you mean? Seeing just RNA on your gel after the entire RT-PCR procedure? Seems unlikely as RNA is very unstable at higher temperature and you're going up to 95 for a total of several minutes in most programs...
Do you have a positive control set of primers (constituitively expressed gene)?
I would check that...if that doesn't come through, something is wrong with your RNA prep.
Change your kit!!try RT PCR Kit fromABI
I've used this kit exclusively for end-point RT-PCR and it has always worked for me. Are you using sequence specific primers That is the only way I have done it and never had a problem. Just find a good annealing temperature. I usually use an applet from any of hundreds of website, but make sure it uses the nearest-neighbor method for your primers. If you are using oligo dT primers, I've heard that they can have lower efficiency. Other than that Iwould check on the usual suspects for normal PCR.