Diagnostic Restriction Mapping Of The Plasmid Construct With NcoI-HF

April 30, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

NcoI-HF

Catalog Number:

R3193S

I have constructed a plasmid construct called pCU190_dgp of a size approximately 19 kb by Gibson assembly method. This plasmid prepped from carrier E.coli cells was subjected to diagnostic restriction mapping with NcoI-HF. Digestion with NcoI-HF was expected to give 4 DNA fragments. 1% agarose gel run of digested plasmid preps revealed expected restriction pattern as seen in 1% agarose gel attached.

Experimental Design and Results Summary

Application

Diagnostic restriction mapping of the plasmid constructs and molecular cloning

Starting Material

1 microgram of the plasmid DNA

Protocol Overview

Restriction digestion was performed as a 50 microliter reactions with 1 microgram of plasmid DNA prep, NEB's Cutsmart buffer to a concentration of 1X, and 1 microliter of NcoI-HF enzyme. Digestion mix were incubated at 37 degree C for 30 minutes. Then reactions were stopped by adding 6X DNA loading dye and analysed in 1% agarose gel.

Tips

Plasmid DNA preps should be of high quality and free from high salt contamination.

Results Summary

Restriction digestion of pUC19_dgp with NcoI-HF gave 4 different sized DNA fragments of expected pattern as seen in 1% agarose gel.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy and simple procedure. Time saver qualified and high fidelity enzyme in restricting.

The Bad

None

The Bottom Line

Diagnostic restriction digestion of the my plasmid construct (pUC19_dgp of size 19 kb) went well and confirmed presence of constructed plasmids in the transformed E. coli cells. I recommend this enzyme from NEB for cloning and diagnostic restriction mapping of the plasmids if they have NcoI-HF restriction sites.

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