Diagnostic Restriction Mapping Cloned Plasmid Constructs From E.Coli Cells Using NotI-HF

May 01, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

NotI-HF

Catalog Number:

R3189S

I have constructed a plasmid called pET-evgS of size 7.12 kb. This plasmid was transformed into E. coli DH5 alpha cells and selected on agar plates with selective antibiotic marker. Selected clones were then tested for the presence of the plasmid by prepping plasmid and subjecting it into restriction digestion with NotI-HF which will give 4 DNA fragment of sizes 4.3, 1.5, 0.82 and 0.5 kb. Indeed, all the clones tested gave expected restriction pattern and confirmed the presence of the pET-evgS construct.

Experimental Design and Results Summary

Application

Restriction cloning and diagnostic restriction mapping of the plasmid constructs

Starting Material

1 microgram of the plasmid DNA

Protocol Overview

Restriction digestion was performed in a 50 microliter reaction volume with 1 microgram of plasmid DNA, appropriate NEB buffer system (cutsmart buffer), and 1 microliter of NotI-HF enzyme. Reaction mix was incubated at 37 degree C for 15 minutes. Then reaction was analysed in 1% agarose gel run.

Tips

Plasmid DNA should be pure and free from any salt contamination.

Results Summary

Restriction digestion of the plasmid construct pET-evgS gave DNA fragments of 4.3, 1.5, 0.82 and 0.5 kb, which is an expected restriction pattern with XhoI. This confirmed the presence of my plasmid construct in the E. coli DH5 alpha cells.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy and simple protocol. Time saver qualified and digestion can be completed within 15 minutes.

The Bad

None

The Bottom Line

XhoI digestion of the plasmid prepped from E. coli DH5 alpha cells transformed with pET-evgS plasmid construct confirmed the presence of the plasmid. All clones tested revealed expected diagnostic restriction pattern.

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