Long pcr frustration and problem.. all help is Much appreciated - (Sep/29/2017 )

I have once managed to amplified a 6.5 kb pcr.
However, the amplification is not reproducible again using the same pcr condition, cDNA samples, primers, high fidelity enzymes. Instead of getting the intended product, a band of less than 2kb base pair is obtained. I?ve tried reextracting the rna, redo reverse transcription, changing dntps, nucleuse free water, new aliquote of primers, new aliquote of enzyme and buffer, but still nothing seem to work. I appreciate All advices to reproduce the 6.5 kb product as it is crucial for my downstream. Thanks a million.

So - what are the conditions you are currently using?

Hi bob1.
I am currently using neb q5. The pcr cycle is as follow,
98c, 30 sec initial denaturation
35 cycles:
98c, 10 sec
67c, 30 sec
72c, 7 mins
Final extension 72c, 2 min
dntp final conc. 200 microm, forward reverse primers 0.5 microm each, polymerase 0.02 u/ microl
It once worked getting the intended product, but things went off after that.
I?ve also tried the gc enhancer but still a less than 2kb product is obtained regardless of running a gradient.
I appreciate your advice.

How much template are you adding? You typically need 10-100 ng of cDNA for a 50 ul reaction.

I think Q5 requires more Mg2+ than regular PCR - consider adding up to 1 mM more than total dNTP concentration.

I would use a shorter annealing time in the cycling step - 30 s should be enough. The final extension should be the long one - basically here, all you are trying to do is provide a start to the extension, which will then be finished off with the longer extension. 5 min should be enough - 40-50 s/kb generally.

Have you tried an annealing temperature gradient?

You might want to check the integrity of the RNA - use a bioanalyzer if you have one, or run a bleach or formamide gel.

Try adding some DMSO or Betaine to the reaction to prevent secondary structure formation.

As you have a long template you might want to increase the initial denaturation time to 2-3 min.