I'm trying to clone some cDNA, but when I PCR the cDNA with my primers, I get no product on the gel. So I checked around and found out that I might be missing the second strand synthesis step, could this be the reason for the lack of product? My cDNA is fine, and appears on agarose gel, and I have also used this for qPCR, amplicons look fine on the bioanalyser.
Also the second strand kits are too expensive to purchase, does anyone have a protocol that has worked for them? I can purchase the second strand buffer from NEB, they used to sell this with the second strand enzyme mix (RNAse H, E coli polymerase and E coli ligase) but have discontinued it.
My second issue is that the e coli ligase in the quantity on the NEB protocol is a lot and also too much to purchase, does anyone have any protocol without e coli ligase? Will be very grateful for your help, been stuck on this process for too long. Thanks
Abiola,
second strand synthesis is not required to do a RT-PCR but
I don't understand why your qPCR is working but not your rt-PCR (maybe your PCR is amplifying the genomic DNA?) if your cDNA is not too long try RT with random priming then PCR with the specific oligos add restriction sites on your oligos (that are not in the sequence you want to clone of course) and clone the PCR product after digestion....
As Didier Poncet pointed out, you should be able to do PCR with only one cDNA strand. And if your qPCR is working, then that confirms it. I would double check the PCR conditions, are your primers really correct and expected to be present on the cDNA? Do you have the right conditons?
Dear Abiola,
during PCR, the forward primer will be used to synthesise the second strand thus allowing you to have both strands for PCR, so, as Michael and Didier suggested, the problem is not there. However the retrotranscription kit you use might influence the outcome: some kits are optimised to obtain cDNA in small size to perform qPCR (where you usually amplify small amplicons), but you may not retrotranscribe enough full length template to be able to efficiently amplify the ORF. Especially if the ORF is large!
Thus, i suggest that you check the characteristics of the reverse transcriptase kit you are using. You could use the specific reverse primer you are using for PCR amplification also for reverse transcription, instead of oligo dT and random primers. This would increase the yield of your gene of interest. Then, of course, you may have to optimise also PCR conditions and allow enough time for extension. Good luck!
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