High-Fidelity and Robust PCR for Difficult DNA Insert Amplification

February 07, 2020

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

New England Biolabs

Product Name:

Phusion High-Fidelity PCR Kit

Catalog Number:

M0531S

Using Phusion High-Fidelity PCR kit, I was able to successfully amplify difficult amplify DNA insert which had greater than 65 percent GC content. I used GC enhancer which comes with the kit and followed optimization protocol mentioned in the kit. 5 Kb DNA insert was sucessfully amplfied for further cloning and its sequence was verified my sanger sequencing which revealed no muations in the amplified DNA insert.

Experimental Design and Results Summary

Application

High Fidelity and robust PCR for cloning

Starting Material

50 ng of Donor DNA template (which had greater than 65% GC content) per 50 microliter reaction

Tips

The kit comes with GC enhancer buffer for amplification of the DNA inserts with high GC content. I used the GC enhancer to improve PCR performance

Results Summary

The PCR was optimised using different gradient temperature and using GC enhancer buffer which comes with kit. PCRs were performed in a 50 microliter reaction volume with 50 ng DNA donor template. The protocol was followed exactly as mentioned in the kit's instruction manual. After PCR, few aliquots were analysed by 1% agarose gel electrophoresis and found that expected PCR product (5 Kb). Remaining aliquots were column purified and used for further coloning into a plasmid. After cloning, the insert part was verified sanger sequencing. sequencing revealed no errors/mutions.

DOI or PMID #

N/A

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Summary

The Good

High fidelity and PCRs can be optimised quickly. It has 50 times high fidelity over Taq polymerase

The Bad

None

The Bottom Line

This PCR kit is very ideal if one has difficult to amplify template DNA like those DNA insert which have more than 60 % GC content.

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