I have a rather tricky PCR that is not working out. I had previously ruled out the kit itself through positive controls, grown new template DNA with a miniprep, changed my water and tips and anything else I could think of.
I submitted the primers and two different templates with the target gene for sequencing, expecting them to come back negative. Of course, they came back with a positive result today, leaving me puzzled as to what could be going on.
Retrying PCR left me with a completely blank agarose gel, same as before.
Here are the primer sequences. I'm trying to insert very standard RE sites, NheI and BamHI, respectively, represented in caps, into areas where SalI and SwaI used to be, and trying to clone out the EF1a promoter in the stock plasmid.
Fwd: 5’ t cgatGCTAGC gataagcttt gcaaagatgg 3’
Rev: 5’a ttaaGGATCC gtcgaaat tcctcacgac ac 3’
Also, the addgene sequence page for the template: http://www.addgene.o.../sequence/7269/
My only thoughts are that there may be some heterodimer interactions going on between the two, but the dG values of the calculated heterodimer interactions are comparable to PCRs that have worked before, according to IDT (http://www.idtdna.co.../OligoAnalyzer/)
I appreciate any thoughts or ideas, as well as prayers and small donations. Thanks in advance.