We are going to do real time PCR with TaqMan SNP Genotyping assay to determine the genotypes of a population. However we donot have a positive control. I am wondering that is it possible to do sequencing after real time PCR to confirm a sample is heterozygote or homozygote? Is there any way to confirm the results of a TaqMan kit for real time PCR?
Of course you can sequence your Taqman product, just like any other pcr product. Just clean up your product and run your sequencing pcr. If your sequence can be found in BLAST you will get a hit.
Of course we know the sequence. I just want to confirm the results. Probably they are okey but we donot have positive control sample. I didn't be sure this sample is definitely homozygote for allele 1, for example. So I thought that is it possible to sequence the RT-PCR product to confirm the results. Is it possible to confirm results in any other way or I just have to trust the TaqMan and RT-PCR design and have to think they've already confirmed by probe. I think I just wonder that how the kit manufacturer be sure about this issue 100%.
I didnot see difference btw negative control and my sample. I think everything is okey and works well. Thank you for help :)
Of course you can sequence your Taqman product, just like any other pcr product. Just clean up your product and run your sequencing pcr. If your sequence can be found in BLAST you will get a hit.
Dear Ali,
I donot have any problem with RT-PCR or TaqMan Assay or results. Just I want to check the products. I will try the sequence them.
Hello Sinem,
Another way of checking whether your product is homo- or heterozygote is a melting curve analysis if your cycler can do that. Probes are perfect for this.
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