Diagnostic Restriction Mapping Of The Plasmid Construct With PvuII Enzyme

April 27, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

PvuII

Catalog Number:

R0151S

I have constructed a plasmid called pUC19_Pgl2 of a size approximately 6.95 kb and transformed it into E.coli cells. After transformation, colonies were picked and prepped for plasmid by plasmid miniprep kit method. Presence of correct plasmid in the transformed cells were confirmed by diagnostic restriction mapping using NEB's PvuII enzyme. Restriction with PvuII is expected to give two DNA fragments of size of 6.5 and 0.45 kb. Indeed digestion of pUC19_Pgl2 with PvuII gave expected restriction pattern as seen in 1% agarose gel.

Experimental Design and Results Summary

Application

Diagnostic restriction mapping of the plasmid constructs

Starting Material

1 microgram of plasmid DNA

Protocol Overview

Extracted plasmid DNA preps were digested wit PvuII enzyme in a 50 microliter reaction volume with 1 microliter of enzyme, 1 microgram of plasmid DNA, and NEB's Cutsmart buffer to a final 1X concentration. Reaction mix were incubated at 37 degree C for 1 hour. Then analysed in 1% agarose gel run.

Tips

High quality plasmid DNA preps are important. Should be free from high salt contamination.

Results Summary

Restriction digestion of pUC19_Pgl2 plasmid construct with PvuII gave two DNA fragments of size 6.5 and 0.45 kb as seen in 1% agarose gel run.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy and simple to perform protocol. Not an expensive enzyme to buy.

The Bad

None

The Bottom Line

Diagnostic restriction digestions of the plasmid preps (pUC19_Pgl2) with PvuII went well and happy with the results. I recommend using PvuII enzyme from NEB for cloning experiments if the plasmid constructs have PvuII restriction sites.

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