Restriction Cloning And Diagnostic Restriction Mapping Of Plasmid Constructs

April 13, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

SacII

Catalog Number:

R0157S

I have constructed pET30 plasmid with my gene of interest. Transformed this plasmid into suitable E. coli cells. Quickly wanted to check whether my clones have pET30 construct and used NEB's SacII restriction enzyme to check correct plasmid being transformed and propagated. SacII restriction expected to cut at two sites and yield two bands of 5692 and 796 bp. Indeed my plasmid preps digested with SacII enzyme gave correct restriction map as analysed by agarose gel run.

Experimental Design and Results Summary

Application

Restriction cloning and diagnostic restriction mapping of the plasmid constructs.

Starting Material

1 microgram of plasmid DNA from E. coli cells in a 50 microliter reaction volume.

Protocol Overview

Perform restriction reactions in a 50 microliter total reaction volume. Use 1 microgram of plasmid DNA with appropriate buffer. I used 10X Cutsmart buffer to a 1X final concentration. Then add 1 microliter of SacII enzyme, mix well, incubate at 37 degree C for 2 hours. Then perform 1% agarose gel analysis.

Tips

Use appropriate NEB buffer which compatible with enzyme.

Results Summary

Restriction digestion with SacII on my plasmid preps gave expected restriction map of 5692 bp and 796 bp.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy to follow protocol and not an expensive buy.

The Bad

None

The Bottom Line

Excellent source of SacII enzyme from NEB. Restriction digestion went well using Cutsmart buffer for my plasmid constructs having SacII restriction site. Highly recommend this enzyme.

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