To get a gene's full sequence, I design some primers according to the RNAseq result and cDNA-RACE. it should be note that RNAseq result is consistent with cDNA-RACE result. i try to make the primer F and R target to the fore and aft ends (ATG and TAG), respectively. however, those primers either could not result any stripe or the stripes turn out to be irrelevant sequence.
what's wrong with my PCR?
it drive me nuts.
What is your source of genomic DNA? Depending on the source, you could possibly have intronic sequence present and the distance between your F and R primers is simply too large for your enzyme. I would suggest an XL PCR to try and get it amplified.
Have you done any analysis on the designed primers such as identification of hairpin, self-dimer and heterodimer formation?
If the formed bands/stripes are way smaller than the desired size, then it is likely that the primers are not doing their proper job.
In terms of PCR, tuning the annealing and extension temperature may help the primers to anneal.
Hope this help. Best of luck.
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