PCR band moves lower than its size on agarose gelsecondary structure
Posted 14 August 2012 - 07:36 PM
I have amplified a 3.5 kb band with PCR but on agarose gel it stops at nearly 3.1 kb. I gel extracted the band and ligated it to pJet and sent for sequencing. The sequencing results show this is the same 3.5 kb band that I was expecting.
But then again, when I digest the plasmid with RE enzymes to get the insert out, the band that appears is exactly at the same size of 3.1 kb. It's driving me mad. All other PCR products move to 3.5 kb, except this product.
Posted 14 August 2012 - 11:44 PM
Posted 15 August 2012 - 09:28 PM
Posted 16 August 2012 - 04:11 AM
Also differences in other reaction components maybe causing shift, as seem commonly in the RE digest bands. We add appropriate restriction buffer to our DNA marker when checking restricted fragments. Doesn't happed in PCR though, but maybe there is something in your reaction that makes it more charged.
I never trust anything that can't be doubted.