Diagnostic Restriction Mapping Of Plasmid Construct With BglII Restriction Enzme

April 24, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

BglII

Catalog Number:

R0144S

I have constructed a plasmid called pCVD442_operon B of a size 18992 bp size. This plasmid was prepped from carrier E. coli strains and digested with BglII restriction enzyme from NEB. Restriction mapping was expected give 6 bands of DNA fragments on 0.7 % agarose gel run. Indeed, my plasmid preps tested with diagnostic restriction using BglII gave 6 bands as expected (seen in attached gel picture).

Experimental Design and Results Summary

Application

Diagnostic restriction mapping and cloning of the plasmid constructs

Starting Material

1 microgram of plasmid DNA

Protocol Overview

Restriction digestion were conducted in a 50 microliter reaction volume with 1 microgram of plasmid DNA prepped from E. coli cells, NEB's Cutsmart buffer to a 1X concentration and 1 microliter of BgIII enzyme. Reactions were incubated for 1 hour at 37 degree C. Then reactions were stopped by adding 6X DNA loading dye and analysed in 0.7% agarose gel run.

Tips

If you are working with large plasmid constructs, I suggest prep them using plasmid midi prep kits. Also make sure the plasmid DNA is free from any salt contamination.

Results Summary

Restriction mapping with BglII enzyme on pCVD442_operon B plasmid construct gave expected restriction map (6 different DNA fragments) as attached in the 0.7% agarose gel.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy and simple protocol. Digestion can performed within 30 minutes.

The Bad

None

The Bottom Line

Very satisfied with using BglII restriction enzyme from NEB for diagnostic restriction mapping of the plasmid construct pCVD442_operon B. Bgl II digestions performed very well with my plasmid constructs.

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