Amplification of DNA by PfuS

For cloning of pieces of DNA, use Pfu or better PfuS enzymes for amplification. They contain proof reading activity and have a high processivity. For analytical purposes of short/medium sized DNA, you may use Taq, which has a better amplification efficiency.
Compared to Pfu, PfuS is faster and has a higher processivity. This allows you to amplify DNA with even more than 5000 bp length. It is comparable to the LongRange kit of Roche. In addition, the PfuS enzyme mix contains pyrophosphatase, which significantly increases the reaction yield.

Pipetting scheme:

Component volume volume final conc.
(50 µl mix) (20 µl mix)
water fill up fill up
5x buffer 10 µl 4 µl 1x
10 mM dNTP mix 1 µl 4 µl 200 µM each
primer A (10 µM) 2.5 µl 1 µl 0.5 µM
primer B (10 µM) 2.5 µl 1 µl 0.5 µM
template DNA * *
DMSO 1.5 µl 0.6 µl 3%
PfuS mix 0.5 µl 0.2 µl 0.02 U/µl

5x buffer: 5x Phusion HF buffer from Fermentas
DMSO: is optional
template DNA (per 50 µl mix): low complexity DNA (e.g. plasmid, BAC) 1 pg to 10 ng genomic DNA 250 ng


PCR programme

No. StepTemp. ¡C Time
1 Init. denaturation98.5 30 s
2. Denaturation98.5 10s
3 Annealing* 10-30 s
4 Extension72 15-30 s per 1 kb
got to step 2, repeat 25 to 35 times
5 Final extension72 5 min

Annealing temperature: For primers > 20nt, anneal for 10 30 seconds at a Tm +3¡C of the lower Tm primer. For primers with less than 20nt, use an annealing temperature equal to the Tm of the lower Tm primer. The TmÕs is calculated with the nearest neighbor method.



PfuS enzyme mix

The enzyme mix consists of three enzymes, that are each recombinantly purified. Please take a look at the section "Recombinant proteins" for a detailed description of the purification and background.

DNA polymerase PfuS (101.9 kDa) PfuS is a derivative from Pfu Polymerase, which has proofreading activity. It is very similar to Phusion polymerase.The additional DNA binding module of PfuS increases DNA binding. Therefore a denaturing temperature of 98.5¡C is strongly recommended; otherwise the Polymerase will not be completely released from the (old) template DNA.
Pyrophosphatese (21.9 kDa) It can convert pyrophosphates to phosphates. Pyrophosphates accumulate during the dNTPs incorporation into the new synthesized DNA strands, which will hinder the PCR reaction under the high concentration of pyrophosphates. Pyrophosphatese can increase the yield of PCR products by converting pyrophosphates to phosphates.
dUTPase (20.4 kDa, but runs at 18 kDa in SDS-PAGE) It is an enzyme that hydrolyzes dUTP, which may be a contamination within dCTP and can be a thermal desamination product from dCTP. If dUTP is present during PCR and incorporated into DNA, Pfu or PfuS polymerase binds nearly irreversibly to this site and thus poisons the reaction. dUTPase can overcome dUTP incorporation by hydrolyzing dUTP.