the important thing in long pcr is that the template does not re anneal to itself before the extension happens so all long pcr should have primers that anneal at very high temperature to minimise the time that the template has to become double stranded again. I would design primers annealing close to 72c and do the pcr in the presence of 1Molar betaine which keeps the template melted for longer by destroying the triple hydrogen bonding of C-G bonds so makes the template melt easier. A profile of 95c,70c,72c would work much better . 53c is very low for long pcrs

Try using DMSO in you reaction it will help if the DNA fragment which you wish amplify has higher GC%. 

the important thing in long pcr is that the template does not re anneal to itself before the extension happens so all long pcr should have primers that anneal at very high temperature to minimise the time that the template has to become double stranded again. I would design primers annealing close to 72c and do the pcr in the presence of 1Molar betaine which keeps the template melted for longer by destroying the triple hydrogen bonding of C-G bonds so makes the template melt easier. A profile of 95c,70c,72c would work much better . 53c is very low for long pcrs

I agree with Paul. If you are still having problems, I think you have no choice but to break your desire product into smaller pieces and stitch them together after all the amplifications. And also, check if your desire region has some parts with very high GC content. If so, I think you would have to deal with those parts differently (use phusion with high GC buffer, touchdown pcr from high temperature, order oligos and anneal them together etc.).

Good luck.

Thank you for your valuable points. I am agree with you. 53c is very low for long pcrs. My primer length is 28 bp from start codon and this part does not have high GC content. So primer anealing temp in best condition is 53 to 55. On the other hand I need to amplify full length CDS for gateway cloning. Thus I cant design primer in any other part of CDS. Do you have any idea to solve this?

Hello Ramin Bahmani,

did you try to reduce your DNA matrix quantity?

I had a similar problem when I was amplifying DNA fragment with high GC content (around 85% of GC) but with a length of 1.5kbp. However reducing the quantity helped me to eliminate the smear and keep the wanted fragment. I had to use 100 pg of DNA matrix.

hopefully this could help you.

Good luck

I don't see the reason why you cannot design primers targeting other parts of the CDS. Yes, you are trying to get the full length product. Designing primers targeting other parts of the CDS to break your 5k product into five 1k product and ligate them together can also give you the full length product (I know it's very painful but sometimes you just have to do it).

But before you do that, maybe you can try doing a touchdown pcr. What I usually do is to start at 70 degree annealing temp and gradually go down to 60 degree with 3 cycles per degree and then do 15 cycles of 58 degree annealling temp because my primers are usually 58 to 60 degree annealing temp.

And again, good luck buddy.

Jack is right and I am sure that your primers will anneal at a higher temperature than the calculated annealing temperature. Why not just make your primers longer by adding bases at the 3' end to raise the annealing temperature. If they show some homology at the 3'end so you are worried about primer dimer problems then use a hot start enzyme which makes p-d very difficult to form or just do not add enzyme until the reaction mix is at high ( >70c) temperature and add your enzyme in an appropriate volume to the hot mixture. Enzyme mixing will happen by thermal diffusion. if you have a gradient pcr machine it might be worth trying a gradient of annealing temperatures because  I am sure that 28mers will anneal at a higher temperature. The Tm is calculated for 50% annealing and a lower percentage in the early cycles will still work even if a couple more cycles are required for a visible amount of product

By using modified nucleic acid you can increase annealing temperature of your DNA fragment. this will help your primer to work efficiently and hopefully will help in solving your problem.

Actually Amplifying 5kb will not be a issue by using Regular Process, i suggest you to reduce the initial denaturation time i.e., from 5 minutes to 3 minutes

 Initial denaturation time can be reduced to 30 sec. Denaturation time in each cycle can be reduced to 15 sec. 30 cycles is enough. 5 kb template requires 3 min elongation. I was always quite satisfied with Roche Pwo master mix. Taq:Pfu(Pwo) 30-50:1 sometimes gives better results. If the template is a plasmid, it should be properly diluted (

NO tips, unless you use the high-field polymease, but the mutation also exist ,people can not control but use better polymease to less miss

I would also try to increase the temperature from 53 to perhaps 58 to start with... you said you tried gradient PCR. what annealing temperatures did you set up? Sometimes you can add an enhancer, that worked for me. Just try to modify conditions of your PCR one at a time. Good luck!

Thank you for all comments!

Special thanks to Paul. Finally using the Touchdown PCR method and increasing the primer annealing temperature I amplified my gene of interest.