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PCR Help - 2kbp product - (Feb/19/2008 )

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Well, I have 3 sets of primers all 55% GC, 60C Tm, BLASTed all primers, checked secondary structures, surely two should work together smile.gif, I'll look into Triplemaster as well.


QUOTE (vetticus3 @ Feb 22 2008, 03:15 PM)
have you looked at the primers themselves... not where they bind.
Are they GC rich?
What are their temps?
Do they form stem loops etc?
Perhaps you just need new primers that are better suited.

If it is the polymerase, i'd also like to suggest Triplemaster.
Triplemaster is fantastic (just PCR'd up 7.4 kb with that).



QUOTE (MKR @ Feb 22 2008, 02:08 PM)
I want to sequence the products, so I do want fidelity...right? smile.gif I'll try KODhifi.

If you want to do population based-sequencing of PCR product you don't need high fidelity (consider you have 10 template molecules and say that your enzyme makes a mistake in 1 product out of 10 in the first amplification cycle at a specific location, you should theoretically have that error in 10% of your sequencing reaction which is beyond the detection limit of standard sequencing).

If you need to do cloning and sequence clones, or if you'll do pyro sequencing: go for as high fidelity as possible.


Don't know if you're still considering raising the denaturation temp, but if you are, don't, unless the enzyme you choose specifically states you can go that high.
Taq is great at 94 degrees. But increase that to 95, and the half life plummets. You'll never get through an experiment.


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