Diagnostic Restriction Mapping Of The Plasmid Construct With XhoI

May 02, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

XhoI

Catalog Number:

R0146S

I have designed and constructed a plasmid, pET20-cadA of size 6130 bp. After transforming this plasmid into E. coli BL21 cells, positive clones were selected and the plasmid was prepped. The prepped plasmid was subjected to diagnostic restriction mapping using XhoI to confirm the presence of the plasmid in E. coli BL21 cells. XhoI restriction will give two DNA fragments of size 4275 and 1855 bp. Indeed plasmid plasmid prepped from 4 clones revealed correct restriction map as seen in 1% 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 prepped using Qiagen plasmid miniprep kit

Protocol Overview

Digestion of the plasmid prep was carried out in a 50 microliter reactions with 1 microgram of plasmid DNA, NEB's Cutsmart buffer to final concentration of 1X, and 1 microliter of XhoI. Reaction mix was incubated at 37 degree C for 1 hour. Then analysed in 1% agarose gel.

Tips

Plasmid DNA should be free of any high salt contamination.

Results Summary

Restriction of the plasmid, pET20-cadA construct with XhoI gave DNA fragments of 4275 and 1855 bp as seen in 1% agarose gel run. This was expected restriction map and confirmed presence of the plasmid constructs in the E. coli BL21 clones tested.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy and simple protocol to perform

The Bad

None

The Bottom Line

Restriction digestion of the plasmid construct went well and gave expected restriction pattern. Tested clones were confirmed for the presence of correct plasmid. Overall satisfied using this enzyme.

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