Amplification Of Gene Templates For Gibson Assembly Cloning

July 20, 2016

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

Q5 High Fidelity DNA polymerase

Catalog Number:

M0491G

I used Q5 High fidelity DNA polymerase from NEB for generating PCR templates to assemble them in plasmid expression vector using Gibson assembly reaction. When I used Taq DNA polymerase, I was getting error prone template sequences and frequently failing in Gibson assembly reaction. Then I switched to Q5 polymerase and good quality templates to use in Gibson assembly cloning and it worked very well. I checked sequences of template after Q5 polymerase PCR in Sanger sequencing method and found that template sequences were without error. I recommend doing PCR with Q5 polymerase if you want to perform HiFi assembly or Gibson assembly cloning.

Experimental Design and Results Summary

Application

Amplification PCR templates for Gibson assembly cloning

Starting Material

5 ng total genomic DNA of Salmonella enterica

Protocol Overview

50 ng total genomic DNA of Salmonella enterica was used as template in 50 microlter reaction volume using Q5 high fidelity DNA polymerase to amplify phoQ gene. I followed standard protocol mentioned in the Q5 high fidelity polymerase kit. At the end of reaction, measured DNA concentration using Nanodrop ND1000 spectrophotometer.

Tips

If your target template to be amplified has high GC content, then use 5X High GC buffer which comes with this kit.

Results Summary

After PCR, I got good yield and quality as checked by Nanodrop ND 1000 spectrophotometer (260:280 ratio of my templates were 1.85 to 1.87 with 190 ng/microlter quantity). I also checked the sequences of the template by sanger sequencing method and were looked without any errors in the sequences. Then used this template to assemble in pBAD vector using Gibson assembly method and assembly reaction worked well.

Additional Notes

None

Image Gallery

Summary

The Good

Easy-to-perform protocol. You will get high quality PCR templates for cloning reaction.

The Bad

None

The Bottom Line

If you are on to performing high quality cloning assembly like Gibson reaction, then go for amplifying your assembly templates using NEB's Q5 high fidelity DNA polymerase.

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