Amplifying Large AT rich amplicons with Pfu type polymerases

This is a protocol for amplifying large amplicons that are high in AT. I developed it in order to amplify the 8.2 Kb region containing the promoter of Arabidopsis FT which is approximately 70% AT. In my hands it worked reliably with an 8 Kb amplicon (DeBono notebook 1, July 2010) but can be easily modified for longer products using more dNTP and optimized template concentration.

Methods

1. Very clean, homogeneous DNA. This protocol may work with genomic DNA but it is best to use DNA that contains many copies of your desired amplicon such as a BAC or a plasmid. I was using ~ 120 ng of BAC DNA.

2. A Pfu-like polymerase marketed as iProof, Phusion, Q5, and KAPA HiFi.

3. Low temperature 2-step PCR. 2-step PCR excludes the annealing step. The primers anneal to the template as the temperature drops to 64.5oC. Extension also occurs at 64.5oC.

Here is my 2-step PCR cycle:

98oC 60 s

98oC 20 s

64.5oC 90 s/1 Kb

Repeat steps 2-3 30-40 times

Polishing step at 64.5 for the amount of time specified in step 3.




This PCR regimen worked well for the Arabidopsis FT promoter as well as amplifying recalcitrant pectin methylesterases.

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