Amplification Of High Quality DNA Fragment With High Fidelity Using Pwo DNA Polymerase

April 26, 2018

University of Birmingham
Biosciences
Research Assistant

Overall

Quality of Results

Ease-of-Optimization

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Company:

MilliporeSigma

Product Name:

Pwo DNA polymerase

Catalog Number:

11644955001

I have used Pwo DNA polymerase for amplification of 6 kb DNA fragment with high fidelity for cloning into expression plasmid. PCR using this enzyme went well giving good amount of DNA fragment with no errors as confirmed by Sanger sequencing. After purification of PCR product, quality was spot on (260:280 absorbance of 1.88).

Experimental Design and Results Summary

Application

Amplification of high quality DNA for cloning experiments

Starting Material

50 ng of template DNA in a 50 microliter PCR reaction

Protocol Overview

The protocol very simple. I have performed my PCRs in a 50 microliter reactions with recommended specification as given in Pwo DNA polymerase instruction sheet. Only thing, I did is varying annealing temperature to find out best annealing temperature for my DNA fragment to be amplified with primers designed.

Tips

If you are experience poor amplification, try adding MgCl2 with 0.5 mM increments and use high quality DNA template for PCR reaction.

Results Summary

PCR using Pwo DNA polymerase went well. All my cloning experiments using DNA amplified using this enzyme gave right constructs without errors as confirmed by Sanger sequencing.

DOI or PMID #

N/A

Additional Notes

None

Image Gallery

Summary

The Good

High fidelity in amplification when compared to Taq DNA polymerase. Longer half life and more thermal stability.

The Bad

None

The Bottom Line

Cloning constructs using DNA fragments amplified by Pwo DNA polymerase were successfully constructed with out any sequence errors. I suggest this enzyme for PCRs requiring high fidelity.

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