Diagnostic Restriction Mapping Of Plasmid Constructs

April 16, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

BamHI-HF

Catalog Number:

R3136S

I have constructed a plasmid using pAYC184 as backbone with 16 kb operon making it to 20 kb size. I wanted to quickly check the constructed plasmid with restriction with BamHI and SalI double digest. This double digestion is expected give two bands of 16 Kb and 4 Kb. Digestion and agarose gel analysis revealed expected restriction map as seen in agarose gel analysis.

Experimental Design and Results Summary

Application

Diagnostic restriction mapping of plasmid constructs.

Starting Material

1 microgram of plasmid preps in a total of 50 microliter reaction volume

Protocol Overview

The protocol is simple. Perform digestion reactions in 50 microliter volumes with 1 microgram of plasmid DNA, appropriate buffer and 1 microliter of BamHI-HF plus SalI-HF enzyme. Incubate reaction mix at 37 degree C for 1 hr. Then proceed towards 0.7% agarose gel analysis.

Tips

When performing double digestion with restriction enzymes, chose NEB buffer which is compatible with both enzymes. If not, perform sequential digestions.

Results Summary

BamHI-HF and SalI-HF double digestion of the pACYC-operon A plasmid construct gave right pattern of restriction mapping (two bands of 16 and 4 kb) as seen in 0.7% agarose gel run.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy to perform protocol.

The Bad

None

The Bottom Line

Very satisfied with using BamHI in combination with SalI enzyme. Both are time saver qualified and both are compatible in NEB's Cutsmart buffer. Hence simultaneous digestion with both enzymes can be done. Highly recommend this enzyme for use in cloning purposes.

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