New England Biolabs
Phusion High Fidelity DNA Polymerase
M0530S
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I have used Phusion high fidelity DNA polymerase for amplification of DNA inserts for cloning experiments. As a rule of thumb, I wanted to go with thermo-stable polymerase having a 50 fold less error rate compared to Taq polymerase. An added advantage was that it comes with GC enhancer buffer if the template to be amplified is having higher GC content. At the end we got good quality PCR products for cloning experiments.
PCR amplification of DNA inserts for cloning expermients
E. coli genomic DNA
250 ng template DNA was used in a 50 microliter reaction volume with 1X Phusion HF buffer, 500 nM each of forward and reverse primer, 0.2 mM dNTPs mix, 1 units polymerase enzyme. Thermo-cycling conditions were standard as recommended in the protocol except for extension time. The gene which I was amplifying was 3 kb and hence the extension was 90 seconds.
Prepare master mix first for required number of reactions and then dispense individually. Then at the end add your template.
Got good amplification of PCR products with good quality which was seen by sequencing in Sanger method. I found there were no changes in the sequences of the product.
None
Simple and easy-to-follow protocol
Highly recommend this enzyme for generating PCR products intended for cloning methods.
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