PCRing small region on genomic DNA - (May/26/2011 )

Hi, I'm trying to amplify a small (67 bp) region on some genomic DNA samples. I was wondering what conditions I need to consider for the PCR run for amplifying such a small fragment. I've tried the run with an actin control (of ~500bp) and the only thing that shows up on the gel is actin (no other bands are visible).

Currently I'm using 200ng genomic DNA with 400nM primers in a final volume of 25uL. I've run the same sample for RT-PCR using a commercial mastermix and there is product formed (even when run on a gel) so I don't think it is a primer issue.

Any suggestions? Thanks.

Actually, what is your purpose of amplifying of such a small fragment? whay cant you amplify sthg bigger so that is easier to view in gel??

adrian kohsf on Fri May 27 04:08:53 2011 said:

I'm actually going to be doing HRM (high resoution melt) using these primers and for HRM the amplicon size is recommended to be small. But first I want to make sure I'm amplifying the right region so I need to sequence the amplicon. I'm trying to just do traditional PCR first so I can send the sample for sequencing.

HRM amplicons are recomended to be small but not that small. 100 - 200 bp is OK, you may have more problems with amplicon as short as 67 bp. It may be difficult to sequence such small fragment, 30 - 40 bp are usually lost at the beggining and you maybe wouldn't be able to get a clear read up to the end.
If you're not sure what are you amplifying, then your primers may just have wrong sequence and that's the reason why you can't see anything on the gel.