I want to clone a 2kb cDNA fragment from a fungus gene into plasmid pET28a by homologous recombination but I have troubles to amplify full length cDNA. Up to now we have proceeded like this:
1) extraction of total RNA with trizol and chloroform, DNAse treatment
2) RT-PCR with oligo-dT (so at that step we have single stranded cDNA from every RNA that were extracted)
3) amplification of our cDNA of interest (2kb) with phusion and 2 primers to get a double stranded cDNA. The primers I use at that step have a 3' sequence (16 nucleotides) that anneals on the cDNA of interest and a 5' tail (that will add around 20 nucleotides necessary for homologous recombination in pET28a). The problem is my tailed-primers work on genomic DNA but I can't amplify anything from single stranded cDNA.
I have checked with internal primers and I do amplify some fragments (around 200-300 pb) from our cDNA of interest, so I know that my gene is expressed and I have sufficient amount of cDNA to amplify.
So:
- either my cDNA are not complete (they could lack the 3' region for instance since I use oligo dT ?)
-or the primers with tails have troubles annealing on cDNA (and not DNA which seems strange) and I should first amplify my cDNA with specific primers only (that have no tails) ? Or should I use directly a gene specific primer for the RT-PCR step instead of oligo dT ?
thanks a lot for your suggestions!
Dear Margot:
Do you check your RNA quality after purification? Band intensity and ratio between rRNA could give hint of your RNA quality and warrant further molecular work.
Generally the specificity of a primer for PCR amplification relays mainly on the 3' sequence of primer. Generally 20 nucleotides or longer give higher specificity and efficiency in PCR amplification. In RT reaction, oligo-dT primer is continent for preparation cDNA every gene, but it would reduce the relative abundance of a specific gene like in your case. Adapting a mixture of oligo-dT and gene specific primers or gene specific primer, could be options in the next attempt.
If your interested gene containing introns, please check whether your primer pairs in PCR amplification for DNA (not cDNA) amplification are intron flanking. Sometimes, it is very difficult to completely get rid of DNA contamination in RNA preparation even include DNase treatment step. Therefore, at this moment, please ensure the RNA quality first and resynthesis longer primer (without adding sequence for homologous recombination for save money, If amplification successfully, just do 2' PCR using your current primer pair). Also remember to modify your PCR condition if the cDNA is GC rich by adding DMSO or formamine. Hope these would help.
Dear Jrhuau, thanks a lot for your answer. I didn't precise it but of course we checked RNA quality by electrophoresis and it was good. I also did a PCR control on an RNA sample treated with DNAse and there was no amplification as expected, so I'm pretty sure I don't have DNA contamination in my samples. I think it's a good idea to design new primers without adding tails as you suggested! Thanks again!
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