Hi. Nasir

can you kindly mention the PCR program you use? I think you, first check the quality of Your g DNA, then TM of the primers and annealing temperature should be 2c less or equal the TM of the primers, thirdly, the extension time should be not less than 3 min.

Dear Amr,

My PCR program is as follows: Initial Denaturation 95C (10min); Denaturation 95C (1min), Annealing 58C(30sec) and extension 72C(10min) each for 29 cycles; Final extension 72C (10min), storage 4C(infinity). The annealing temperature 2C less than or equal to salt-adjusted Tm calculated by OligoCalc softwere (http://biotools.nubic.northwestern.edu/OligoCalc.html).

Did you use the promega M7122 for long PCR? I need opinion about using this particular kit for long PCR.

Hi, I hope this help

i usually use Vazyme PhantaMax superfedility DNA polymerase Catalog # P505 , I think You can try initial denaturation 95c/5min, followed by 95c/30sec, annealing equal to TM of primers, extension 72c/4min, for 30 cycles then final extension as you mentioned. this conditions works well with me for 8kb from genomic DNA

best of luck

Thank you Mr. Amr. I will follow your suggestions soon!