Hi Abdul,

Jeffrey is right all companies that sell taq will sell a hot start taq and one of your colleagues will have some to test. If not you can do a hot start with any taq by making your pcr mixture with everything except the taq and heat the tubes to 70 c or more. then add a small volume 1-2ul of the taq into the tube with a fresh tip each time and start cycling. This way the mix never gets below about 55c when the enzyme is present so no primer dimer forms. If this works then probably a hot start enzyme is a good idea for your reaction. best wishes, Paul

how many primers have you tried?what's your annealing temperature and your PCR cycle?Please give us more details.

4 types primer i used for it so for. annealing temp. 55, 53 and 50 each individual test. denaturating temp. 95C and extinction temp. 72C. the cycles are 30x and 35x respectivily.

use a hot start enzyme then the primers are never together with an active enzyme at the low temperature needed to produce dimerisation

@Paul Rutland  can you suggest me hot start enzyme? 

@Abdul Majid

There are many hot start enzymes from which to choose. Enzymatics had their Phoenix Taq, Bioline has Immolase along with a couple others, ThermoFisher has several including Platinum Taq, NEB has 2 or 3 including OneTaq. You might find a colleague in your building or complex that already has a hot start enzyme so you might not need to order. In fact, I suggest if your colleagues have to share that you test several as not all will perform the same. Good luck.

Hi Abdul,

Jeffrey is right all companies that sell taq will sell a hot start taq and one of your colleagues will have some to test. If not you can do a hot start with any taq by making your pcr mixture with everything except the taq and heat the tubes to 70 c or more. then add a small volume 1-2ul of the taq into the tube with a fresh tip each time and start cycling. This way the mix never gets below about 55c when the enzyme is present so no primer dimer forms. If this works then probably a hot start enzyme is a good idea for your reaction. best wishes, Paul

and you can tell us what enzyme are you using? and how do you get your DNA template? or you just use the bacteria?

@Paul Rutland Thank you very much Dear Sir. 

@Qing Zhao .. i have used Pfu enzyme mix.. yes i used gDNA  extract from HB8 bacteria.

Pfu enzyme  is a hot start enzyme.so have you done this hot start procedure? or you have already done the procedure as the instruction  protocol of  your enzyme?

i used this enzyme with instruction  protocol as well as change its protocol but didn't get the results, than used anther enzyme taq but the result is same. now i try KOD hot enzyme today see what happened ?

1.are you new in doing PCR and design primers?

2.have you tried  to use the  bacteria instead of the gDNA extract because maybe your template is very rare to amplify the gene?