I am trying to amplify the 10kb fragment using human genomic DNA as template by using the Promega green master mix (M7122) but no result. I am doubtful that polymerase used in this kit might not be able to amplify longer fragments. Did someone use this kit for amplicon size 10kb or more? If yes, then can you please share the optimized PCR conditions? If this kit is not appropriate then can you please suggest the best kit? Thank you
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
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