The length of the construct is 5.6 KB including gene.
PCR reaction set up...
Q5 master mix- 12.5 ul
10uM forward primer-1.25 uL
10um reverse primer-1.25
Template-50 ng
PCR CYCLE-
initial denaturation- 30 sec
16-18 cycle-98 C 10 sec
50-72 C- 30 SEC- 2 Min.
extension-3 minute
final extension 2 minute
Annealing tem. of primer-72C and length 27 base pair
As the length is quite large, I will suggest you to add DMSO (1.5ul of 100% DMSO in 50ul reaction setup) which can help you to get amplification. Also increse the extension time to 5:30 sec and the cycles to around 30. Use denaturation for 2min at 94-98 degree. Good luck.
i would run a gradient of annealing temperatures in case too low a temperature causes non specificity problems between the primers or is allowing the template dna to re-anneal. increase the cycles to 30-35 and increase the extension time to 5 mins per cycle as Pawan suggests
If your problem is with site-directed plasmid mutagenesis, most no amplification is caused by primer-primer dimerisation and low PCR amplification especially with QuickChange primer designing style. You can follow the protocol described in the linked paper which has been proved very efficient. http://www.biomedcentral.com/1472-6750/8/91
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