DNA Insert Amplification for Cloning

January 02, 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:

Q5 Hot Start High Fidelity 2X Kit

Catalog Number:

M0494S

I have used Q5 Hot Start High Fedility PCR Kit to amplify genomic DNA of interest to clone into expression vector. This kit was efficient in amplifying DNA inserts upto 10 kb length without any errors. DNA insert after cloning was sequenced by Sanger sequencing method and found no errors in amplifying 10 Kb length genes.

Experimental Design and Results Summary

Application

Amplification of genomic DNA for cloning

Starting Material

2 ng of genomic DNA from E. coli for 50 microliter PCR

Tips

Use good quality template DNA for efficient PCR using Q5 Hot start High Fedility kit

Results Summary

The PCR reactions were followed according to kit's instructions. The reactions were performed at 50 microliter reaction volume with 2 ng of genomic DNA as template (DNA insert of lenght 10 Kb to be amplified). Extension was carried at 72 degree Celsius for 5 minutes per cylce. The PCR lasted for 4 hrs 30 minutes. After PCR, few aliquots were analysed by 1 % agarose gel and found single band of desiered length. Remaining aliquots were purified and used for cloning.

DOI or PMID #

N/A

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Summary

The Good

Less time on hands as it comes with premixed buffers and enzymes. Efficient in amplifying longer DNA inserts

The Bad

Nothing

The Bottom Line

Highly recommended for amplifying error free longer length DNA inserts. Also, reactions can be set up room temperature.

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