Hello,

I once had a similar problem, I could not amplify a sequence. But, I added 3% DMSO and had an excellent amplified, you can try it.

Best regards

Is your positive control working?

Are these primers for a special purpose (e.g. mutagenesis)? The Tms seem to be quite high for "normal" PCR primers.

Possibly you can try to chose a little shorter primers or introduce a mismatch in the reverse primer to resolve the secondary structure formation.

If the template context is problematic (e.g. very high GC-content close to the target sequence) a restiction digestion of the template might help (given the enzyme doesn't have a restiction site in the target sequence).

How long is the amplified sequence? If it is very long then the elongation time might not be sufficiently long.

You can try a touch-down protocol.

If possible, just design another pair of primers. In the end it often is less time- and money-consuming than trying to make a "bad" primer pair work by tweaking the PCR.

Or you design a different reverse primer for each of your two primers, trying to generate two different but overlapping(!) PCR products. Mixing these products (if you get them) in a combined PCR without adding any further primers will also yield the full-length product (the former products serve as both, primer and template). You can also then try to amplify the full-length product further in a subsequent PCR using the original "outer" primers.

I would add DMSO (final conc. 1-3%) and try a gradient from 50-70°C anneanling temperature. Good luck!

u have to 2 step PCR reaction...if it doesnt works....reduce ur primer length....if possible....

Could you clarify 'strong secondary structure'? is that intentional?, because at the risk of stating the obvious, that's not a good thing..

if it is intentional (sorry, but if it is can I ask why?), then yeah, you would have trouble, but your going to have to work around it...if not, I would suggest redesigning the primers...

If the 3'-end of this primer is not involved in secondary structure formation a considerably lower annealing temp might help.

79-81 is a quite high melting temperature. I choose my designs so that they anneal usually 20 degrees lower.

I would suggest a lower Tm. Try to achieve the Tm around 60 degrees in primer designing and use the annealing from 58-60 degrees. Longer PCR product might give you more control over specificity, but if you are designing for real-time PCR, then try to keep the product size below 200. You can use NCBI website to design or check the specificity of your primer pair (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).

Good Luck

Rohit

I think 2-3% DMSO or betaine or ectoine would resolve your problem.

Hi all, I used 3% DMSO & 2 step PCR too.

Still, did not get he desired band.

What a pity!

If you can be sure that your polymerase is still working, dNTPs are okay (too much thawing and freezing will results in problems) and that your template is there ;) , than now I would not waste to much time in optimizing the PCR. Instead I would design a new pair of primer.

I suggest to re-design the primers with lower Tm (55-65oC). It is very difficult with these Tm.

Thanks everyone. I will instead redesign the primers.

Hi everyone i would like to go ahead with further discussion.

Hello,

I agree with above mentioned comments and redesigning the primers would be good idea. I faced similar problem once, my primers were also forming the strong secondary structure and I could see the primer band at the bottom in the agarose gel. When I used (very less) 0.1µM primer concentration with rest keeping the same, apparently it worked. DNTPs should not be thawed and freezed many times.