Diagnostic Restriction Mapping Of The Plasmid Construct With NheI-HF

April 23, 2018

University of Birmingham
Biosciences
Research Assistant

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

New England Biolabs

Product Name:

NheI-HF

Catalog Number:

R3131S

I have designed and constructed a donor plasmid of size approximately 19.4 Kb for cloning purposes. This plasmid prepped from E. coli DH5 alpha cells were subjected to NheI-HF restriction digestion to confirm presence of the plasmid. Digestion with NheI-HF was expected to give 2 DNA fragments of size 15.9 Kb and 3.5 Kb. Digestion of the plasmid prep with NheI-HF revealed correct restriction pattern as seen in 0.7% agarose gel.

Experimental Design and Results Summary

Application

Diagnostic restriction mapping and cloning of the plasmids

Starting Material

1 microgram of plasmid DNA

Protocol Overview

A total of 1 microgram plasmid DNA, 1X final concentration of NEB's Cutsmart buffer, and 1 microliter of NheI-HF in a 50 microliter reaction volume was set up in sterile nuclease free eppendorf tubes. The reaction mix was incubated at 37 degree C for 30 minutes. Then reaction mix was run in 0.7 % agarose gel at 50 volts for 90 minutes.

Tips

Using appropriate buffer and high quality plasmid preps are critical for successful digestion of the plasmid

Results Summary

NheI-HF restriction gave two DNA fragments of expected size as analysed by 0.7% agarose gel.

DOI or PMID #

N/A

Additional Notes

None

Image Gallery

Summary

The Good

Time saver qualified enzyme. Restriction reaction can be as short as 15 minutes. Easy and simple protocol.

The Bad

None

The Bottom Line

Restriction mapping of my plasmid construct (19.4 Kb) with NheI-HF enzyme performed very will within 30 minutes of digestion. I recommend using this enzyme on plasmid constructs having NheI-HF restriction site.

Join the discussion