PCRing small region on genomic DNA
Posted 26 May 2011 - 03:19 PM
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.
Posted 26 May 2011 - 08:08 PM
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
Posted 27 May 2011 - 11:59 AM
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.
Posted 27 May 2011 - 12:11 PM
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.
I never trust anything that can't be doubted.