RF-Cloning.org Forum

rfuser

Member

Registered: 2021-03-06

Posts: 1

Unspecific binding of primers to target

https://www.rf-cloning.org/rf_cloning_project.php?proj_id=5d42fa53dcd1b8468c681d606a1d0a9e

Hi guys,
I'm trying to design primers in order to amplify only a fragment of a mrna from a cDNA pool and to insert it in a pCDNA. The reverse primer seems great but unfortunately, when I try to blast the forward primer to find other unwanted targets, it finds a lot of cdna which are not the one i'm interested in. I tryed to extend the primer to gain specificity but i don't know if there is a limit in the target annealing temperature.
I hope i explained well the problem
Thanks to anyone who can help me!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Unspecific binding of primers to target

You should be able to get away with up to 70°C.  Generally, you want to keep the forward and reverse primers within a few degrees of one another, so I'd also update the reverse primer as appropriate.
Good luck!
-Steve