Amplification of 25 Kb DNA Insert from Low-Complexity and Difficult Templates from Human Genomic DNA

February 03, 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:

LoAmp Taq PCR Kit

Catalog Number:

E5200S

I have tried several DNA polymerase kits to amplify 25 Kb DNA insert (several genes in cluster) from human genomic DNA for construciton of recombinant plasmid vector but only found success using LoAmp Taq PCR kit from New England Biolabs. I have used 50 ng human genomic DNA as a template in a 50 microliter reaction volume and was able to amplify 25 Kb DNA insert of interest with good quality without any errors.

Experimental Design and Results Summary

Application

Amplification of 25 Kb DNA insert of low complexity from human genomic DNA for recombinant vector construction

Starting Material

50 ng total genomic DNA from human epithelial cells per 50 microliter PCR reaction

Tips

For good and high quality amplificaiton, try several different concentrations of Mg ion supplmentaiton (MgCl2)

Results Summary

Using LoAmp Taq PCR kit, I have successfully amplfied 25 Kb DNA insert of interest from human genomic DNA. Amplified DNA was used to construct a recombinant plasmid vector called pAJ312. The constructed DNA was sequence verified by sequencing and found no mutations in the 25 Kb DNA insert.

DOI or PMID #

N/A

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Summary

The Good

Easy to use protocol and effective in amplifying DNA inserts upto 30 Kb

The Bad

None

The Bottom Line

Ideal PCR kit in the molecular biology labs if one is intending to amplify long DNA inserts from low complexity and difficulty amplify DNA templates like human or eukaryotic DNA samples.

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