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Problems with TA cloning of bisulfite converted PCR product - (Oct/04/2007 )

I'm a third year PhD student and I'm trying to analyze the methylation status of the promoter of a gene.
I have succeeded in amplifying the region of interest after bisulfite conversion but I am having trouble cloning this product.
I have been using the pCR2.1-TOPO vector from Invitrogen TA cloning kit but after sequencing 20 clones only one had the complete 327bp PCR product present. The rest of them had approx 200bp of the product with the 3' end missing. This keeps happening and I can't figure out why.
Any suggestions would be really appreciated.




could there be a TOPO recognition site within your amplicon?



Hello AOG,

So all your clones have insert in them. For those with 200 bp insert, do you see the reverse primer sequence in the insert? Could be that your primers are amplifying two regions of the genome with 327 bp being the expected and the 200 bp non-specific or specific but not expected. You can do a In-Slilco PCR search using your primer sequences (use the sequence for non-converted DNA) to see if you get hits other than the expected.