Diagnostic Restriction Mapping Of Plasmid Constructs

April 03, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

MmeI

Catalog Number:

R0637S

I have constructed plasmid for cloning purpose and it was 4010 bp in size. After transforming this plasmid into recipient E. coli cells, plasmid was prepped from few colonies and checked presence of plasmid by restriction digestion with NEB Mme I enzyme. Digestion with MmeI enzyme will give a single band of 4010 bp on 1% agarose gel. Indeed my all plasmid preps gave a single band of 4010 bp size on 1 % agarose gel.

Experimental Design and Results Summary

Application

Restriction cloning and diagnostic restriction mapping of plasmid constructs

Starting Material

1 microgram of plasmid prep in a 50 microliter reaction volume

Protocol Overview

1 microgram plasmid DNA prepped from E. coli cells in 1.5 ml eppendorf tube along with 1X NEB cutsmart buffer and S-adenoysl methionine supplement to final concentration of 50 micromolar in a 50 microliter reaction volume. Add 1 microliter of MmeI enzyme and mix well by pipetting few times. Incubate the entire reaction mix at 37 degree C for 1 hr. Then stop reaction by adding 6X DNA loading purple dye. Analyse the restriction digested plasmid prep in 1% agarose gel.

Tips

Make sure that your plasmid prep is free of any salt contamination. Salts in plasmid prep greater than 200 mM will inhibit MmeI digestion. Excess MmeI also inhibits restriction reaction.

Results Summary

As seen in 1 % agarose gel analysis, all plasmid preps digested with MmeI at recommended concentrations gave a band 4010 bp, which is expected diagnostic pattern.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy to follow protocol, quick digestion within 15 minutes is possible.

The Bad

None

The Bottom Line

NEB's MmeI restriction digestion with my plasmid constructs worked well and satisfied with using this enzyme. I recommend if anyone want to use this enzyme with plasmid constructs having MmeI restriction site.

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