High Efficiency Error Free DNA Insert Amplification for Cloning

January 25, 2016

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

New England Biolabs, Inc

Product Name:

Q5 High-Fidelity DNA polymerase

Catalog Number:

M0491G

We were regularly using Taq DNA polymerase for amplification of DNA inserts of cloning of bacterial genes in E.coli and used it to get errors in the final DNA insert and low efficiency in cloning. Recently I switched over to Q5 High Fidelity Polymerase for amplification of DNA inserts for cloning. The reason for switching over to Q5 High Fidelity Polymerase is that it can be used for GC rich templates using GC enhancer buffer provided, error free and ease of use. I did check sequence of DNA insert after amplification and found it error free.

Experimental Design and Results Summary

Application

Amplification of evgS gene from E.coli for cloning into recombinant pBAD vector

Starting Material

50 ng genomic DNA

Protocol Overview

25 µl reaction using Kit's instruction and used Q5 High GC Enhance buffer. Cycling conditions as below: Initial Denaturation: 98°C for 30 seconds 98°C for 30 seconds 55°C for 30 seconds 72°C for 30 seconds for 30 cycles Final Extension for 72°C for 5 minutesHold at 4°C

Tips

Quantify DNA template using Qubit DNA quantification kit I used 50 ng total as template for amplification.

Results Summary

After PCR amplification of 1.5 kb evgS gene, got good amplification of evgS gene as clearly visible with attached 1% Agarose gel analysis. Quantification with Qubit HS DNA assay yielded 600 ng/µl. Got sufficient yield to proceed for cloning.Blunt end inserts were generated at the end of PCR.

Additional Notes

None

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Summary

The Good

Good for GC rich templates

The Bad

A bit costlier than Taq DNA polymerase. Otherwise I did not find any drawbacks.

The Bottom Line

Do gradient PCR to determine annealing temperature your primer pair and then accordingly do PCR for desired insert which gives best results at selected annealing temperature.

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