Dear all,
I'm trying to amplify a genomic mRNA of a human gene for Sanger sequencing downstream application. The length of the PCR product is 500pb.
The mRNA are extracted from whole blood samples using TRIzol LS (Invitrogen).
I've tried the RT-PCR two-step (with Hexamer Random Primers, Oligo dT18 and Specific Primers) and One step RT-PCR and I've got the same result, a very weak band of 500pb with other non-specific amplifications.
What do you advise me to get trough this problem and obtain a clear strong PCR product band without any non-specific amplification suitable for Sanger sequencing ?
Thanks
Dear Anass,
Have you checked the quality and quantity of the extracted RNA?
In the case of non-specific amplicons, it's mostly due to non-optimized PCR condition, especially annealing temperature.
Best,
As you are using whole mRNA for amplification you will get multiple amplicons, try to clone expacted one and get it sequenced
Dear Elyas,
Thank you for your answer. Yes I've already checked for the quality/quantity used in the RT-PCR and it's not the problem, since I've also checked the GAPDH gene and I have no problem with the result.
I also tried to resolve the problem by optimizing the annealing temperature by running temperature gradient PCR.
Dear Deepak,
I don't think that cloning will resolve my issue, since I have many samples to work on and sequence. Thank you for responding though.
Dear Anass,
May I ask you how you have designed your primers?
And which Taq polymerase are you using for amplification? Have you checked the specificity of primers you are currently using? Regards,
- Badges
- Science method