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my pcr product is larger than expected - (Aug/20/2012 )

Hello All,

I'm very new to PCR and sequencing, I'm amplyfing a gene of size 560 pb, using published primers, however my product is always larger, I sequenced my product with the same set of primers and I obtained product of 830 bp.

any explanation?


Sequence it using each of your two pcr primers.


Did they work from cDNA and you are working from genomic DNA?


BLAST your sequencing result and you will see what it matches.
You can also check the primers with PrimerBLAST, if you didn't do that already (both genomic and RefSeq mRNA databases) and see if you get something matching your product length.


Just to confirm, is your sequencing result 830 bp long or was the PCR product 830 bp long? As in did you run the PCR product on the gel before sequencing?

-Ameya P-