Neb Builder suggested this primer to me for PCR overlap ligation, and I appear to be stuck with it. At least it isn't 6 guanines! How much trouble do you think this bad forward primer could be?
pcasFrag3_fwd tgttatcgacgatagcaagcttggcggcaacGTGGAGGGCCAAAAAAGAAAAAAG 3'Tm=68.7 3'Ta(annealing temp)=65.5
pcasFrag3_rev taaaatagcgctctcgcgttgcatttctgttcTGTAAAAATGCAGCTCAGATTC 3'Tm=64.5 3'Ta(annealing temp)=65.5
thank you! - Amy J.
I tried to make a loop with CGs but failed so I also think that the polyAs are no problem and neither are quadruplexes ( plices?) so I think that they are fine but at the first hint of trouble use dmso or betaine and any quadruplex will disappear but it should be ok
I find that just a bit of extra Mg and some extra template seems to make low GC % primers work out.
- Badges
- Science method