PCR problem

[ecoli]

I'm having a problem with a gradient PCR I'm running, with the purposed of testing new primers I ordered. all my PCRs are turning up negative results, my positive control is working, though. I'm sure the problem is that my primer has a large overhang, about 70 bp. I've tried annealing temperatures of 45C+ and - 10C, and 55C + and - 10C. The rest of the program is pretty standard. Is there something special I should be doing because of my large overhang?

[ecoli]

part of the problem may be that the overhang is only on one end of the primer.

[ecoli]

Talked to mentor... what he thinks is happening, is that the Taq polymerase found a homolog on the primer, and is binding to it, so it can bind to DNA. What I'm doing, is allowing the primers to bind to the DNA first, then adding the polymerase to the reaction mixture. The PCR is running now, hopefully that worked.

[jeskill]

I'm having a problem with a gradient PCR I'm running, with the purposed of testing new primers I ordered. all my PCRs are turning up negative results, my positive control is working, though. I'm sure the problem is that my primer has a large overhang, about 70 bp. I've tried annealing temperatures of 45C+ and - 10C, and 55C + and - 10C. The rest of the program is pretty standard. Is there something special I should be doing because of my large overhang?

 

 

Just out of curiosity, why would your primer have such a large overhang?

[zyncod]

Just out of curiosity, why would your primer have such a large overhang?

 

I'd agree, especially since most primers have a complimentary sequence of only 30-45 bp. If you've got any restriction sites in your overhang, especially multiple restriction sites, that's going to complicate things because of the stemloops you'll have. If I was you, I would do this in steps, say 20 bp at a time. It's annoying, but I've spent months on a single construct before giving up and doing it the slow, incremental way.

[CharonY]

If such a high overhang is really needed first check if it makes stemloops. What is the calculated annealing temperature for the primers?

What you could also try in theses cases are typically betaine and DMSO.

[ecoli]

I have a his-tag and a linker built into the primer

[CharonY]

I see. Well, if this primer cannot be changed, have you tried another complementary primer and calculate dimers etc.?

If GC is not an issue from personal experience I'd rather toy around with primers (or in some cases with polymerases, e.g. hotstart, or for high GC templates etc.) rather than trying a gazillion conditions.

[ecoli]

my new method didn't work... I'm just going to design a smaller primers and add the his tag afterwards. Oh well. I just wasted 2 weeks on that.