Hey Elton,

I have the solution of your problem. Why did you add the random primer at 3' position. It is very important site for extension. Just add the AT rich flap on 5' position of the forward primer. My most of the research is on it. I can design a flap for you if you will send me the sequences of your primer. Use one of my paper published recently for your reference.

http://link.springer.com/content/pdf/10.1007%2Fs12033-012-9617-5

Let me know if I can help you more.

Cheers!

Why did you design primers with 8 degrees difference in melting temperatures? Which primer tool did you use?

Cheers, Nadine

Its because the forward primer is a specific SL sequence that I am interested. There's no way to change it, and its melting T is very low (62). This is giving me non-specific products. If I try to decrease the Reverse primer it would also give me non-spec bands I guess. So, the best approach would be trying to increase the forward primer..

Hello Elton,

how much Ns did you have? I normally have 24, but some more or less are no problem. So you can try this.

Other possibilities: Put DMSO to the PCR mix, or Q-Solution from Quiagen. Both helps at difficult PCRs. So maybe the combination of these to primers works.

It is better to use gradient from 40-68C. Also what is actual size of your primer. Also you can use DMSO

I've been on the lookout for a way to increase the Tm of primers for some years now, and I'm still looking. There are a few modifications for probes (LNA, MGB) but these don't work well as PCR primers, presumably because the polymerase can't copy them.

I think your best bet with this PCR would be to shorten your reverse primer. 71 degrees is unusually high.

If there is any sequence at all upstream of your primer you may be able to extend into it even if it is quite variable, using G as a universal purine, T as a universal pyrimidine (the G:T basepair is almost as stable as A:T) and inosine as a universal wildcard. Inosine doesn't appreciably increase the stability but it does get you past awkward positions. This shouldn't affect the specificity of your PCR as most of the specificity is steered by the 3' end.

Hey Elton,

I have the solution of your problem. Why did you add the random primer at 3' position. It is very important site for extension. Just add the AT rich flap on 5' position of the forward primer. My most of the research is on it. I can design a flap for you if you will send me the sequences of your primer. Use one of my paper published recently for your reference.

http://link.springer.com/content/pdf/10.1007%2Fs12033-012-9617-5

Let me know if I can help you more.

Cheers!

Random bases should never be added at the 3' end, where Taq starts to extend! Additional bases at the 3' end should be exactly complementary to the target DNA. If the target sequence is known, I would go for this option to increase the Tm of the primer. If you modify the 5' end by adding random bases, you should run few cycles (5-10) of your PCR at a lower annealing temperature to ensure that the primer binds to your target and then go to your optimal annealing temperature for the remaining cycles. You can also try a Touch-down PCR for 10 cycles and then go to your optimal annealing temperature for the remaining cycles.

I'm inclined to agree with Michael. Given the two options he suggests, I lean toward the Touchdown strategy, simply because dipping farther below your optimum Tm always runs the risk of non-specific amplifications...but, having said that, I vote for complete primer redesigns over forcing the issue with the current sub-optimal binding sites. If you can go 'outward a bit' at one end or the other, and still have the functional sequence of interest...I've just never had very good luck with any design where the Tm of the two primers varied by more than about 4-5 degrees...either blah for amplification or the "tons o' bands" effect comes out, neither of which we can typically work with...

NEVER add random nucleotides at the 3' end. You can add whatever you want to the 5' end.

Bob Pergolizzi

Hi Elton,

You may consider to modify your forward primer with a 5' placed TINA (Twisted Intercalating Nucleic Acid) modificaiton?

I believe that by modifying your forward primer with a TINA molecule and maybe also shorten your reverse primer with one or two nucleobases, your primers ought to have quite similar melting temperatures.

We recently introduced this new primer modificaiton, which increase robustness and efficacy of qPCR as well as end-point PCR. Primers are available through Eurofins MWG Operon and the use of modified primers is described in a recent free access publicaiton (pubmed ID: 22701644 ).

Hope that this may help you solve the problem.

Robert is right... add anything at 5' end to control Tm or add restriction site

You should consider adding at the 5' instead of 3' since this is crucial region for binding compare to 5' end.

i think your primer has too high tm.. if you used high tm such 70 or 71, it will not good for reaction. because that t is near the t of polymerase enzyme working... it will better if you can change to new primer with tm not over than 65.

Cheer

as stated by all NEVER mess with the three prime end, try altering/changing the reverse primer if the TM is too high and because the TM is 71 it does not mean you have to run the PCR with the annealing temp that high, it will still work at a lower temp. If your reverse primer has a very high TM perhaps it is too GC rich which may cause a few problems as well.