High-Fidelity Amplification of DNA for Cloning

January 24, 2020

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

Sigma Aldrich

Product Name:

Pwo DNA polymerase Kit

Catalog Number:

11644955001

I have used Pwo DNA polymerase for the amplification of DNA without any mutations where fidelity is of high importance for cloning DNA into bacterial strains especially single molecule DNA inserts been used. This Pwo DNA polymerase gave me high quality DNA after amplification and also confirmed by sequencing which revealed no mutations.

Experimental Design and Results Summary

Application

Molecular cloning and sequencing of genes into bacterial strains

Starting Material

25 ng of recombinant protein producing gene (sodB gene) from a donor plasmid

Tips

If you are using high GC content DNA template source for PCR, then try first different MgCl2 concentration and gradient Temperature

Results Summary

I have amplfied my gene cassette with antibiotic marker from the donor plasmid called pET20b usign Pwo DNA polymerase using 25ng plasmid DNA as template in a 50 microliter reaction. I have followed starndard PCR protocol given in the kit's instruction manual. At the end I have got good quality DNA for further cloning into E. coli genome. Amplfied DNA was of high quality (260:280 spectrophotomer reading was 1.8). Also, the amplified DNA was confirmed by sanger sequencing for any errors and found no erros or mutations in it.

DOI or PMID #

N/A

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Summary

The Good

Simple and easy to do protocol and less hands on time. It has hight thermal stability which is plus if you are doing long hours PCRs.

The Bad

None

The Bottom Line

I recommend this kit for high quality and high fidelity DNA amplification especially planning for cloning important genes and minimise DNA artifacts.

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