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
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??
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.
- hobglobin likes this
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.
'Normal' is a dryer setting. - Elizabeth Moon