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Nonspecific Pfu product cleanup? Should I nest?

Phusion Products Non-Specific Nested PCR Mis-Priming

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#1 BioGeron32

BioGeron32

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Posted 15 June 2012 - 12:48 PM

Hey everybody,

I've been amplifying a largish (7.3kb) amplicon for subsequent sequencing. I'm working off of two genomic templates, and have been experiencing trouble with non-specific product amplification at lower molecular weights (1-4kb range).

Primers:
F: 5'-ATATGAAATTTATTGTGCCCTTTT-3'
R: 5'-TCTGGTCCCGTTTGCTGCTGCCTT-3'

I've blasted these primers to the template for potential mispriming sights. I think I'm simply getting partial products with my enzyme that don't reach full length. Any tips for eliminating these products?

I'm using Phusion pfu polymerase from NEB, in 20uL reaction volumes to achieve a high-fidelity product for later sequencing.

63C TA for 20s, and 3.5min extension time at 72C, 35 cycles total. Everything else is standard for Phusion.

I'm thinking about nesting some of my initial product into a new reaction with a slightly higher annealing temperature to isolate the desired 7.3kb product. I don't think this would compromise fidelity for sequencing, but it is another step away from in-vivo.

Any other ideas? Changes to extension time, additives for the reaction? Anything?

Thanks in advance!





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