Hi all- hope you can help.
I am trying to generate an amplicon library by PCR for sequencing.
My current method is:
1.) First strand synthesis using a biotinylated primer
2.) bind biotinylated product to strep bead
3.) wash bead 2x
4.) second strand synthesis using another biotynylated primer
5.) wash bead 2x
6.) Elute in TE
7) PCR amplicon with universal primers
I am getting the appropriate amplicon size, but i am getting a lot of side product of First strand primer-Second strand primer dimer. This dimer is amplifiable with our universal primer set and no matter how much i wash, i still get enough primer contamination to make this side product.
Does anyone have any fun tricks up their sleeve to get rid of this? I was thinking perhaps instead of biotinylating my primers, i could photobiotinylate my DNA and do my first and second strand synthesis and i could just wash away the primer. Or, do an alternating first strand biotinylation and second strand DIG?
Ideas? Thoughts?...I really need to get rid of this side product
THank you in advance