Restriction Enzyme Mediated Cloning

April 10, 2018

University of Birmingham
Biosciences
Research Assistant

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Quality of Results

Ease-of-Optimization

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Company:

New England Biolabs

Product Name:

BaeGI

Catalog Number:

R0708S

I have used BaeGI enzyme for digesting a donor plasmid (2241 bp size) to get DNA fragment of size 692 bp to clone into another recipient plasmid. Digestion went well and got 692 bp DNA fragment (which can be seen in agarose gel) with good yield and quality after purification of 692 bp DNA fragment from agarose gel.

Experimental Design and Results Summary

Application

Restriction cloning and plasmid mapping

Starting Material

1 microgram of Donor plasmid prep in a 50 microliter reaction volume

Protocol Overview

1. Set up 50 microliter reactions in a sterile nuclease free 1.5 ml Eppendorf tubes.2. Add 1 microgram of donor plasmid prep, then NEB 10X cutsmart buffer to final concentration of 1X in 50 microliter reaction.3. Finally, add 1 microliter of BaeGI enzyme. Mix the contents of the tube by pipetting up and down for 6 times.4. Incubate reaction mix at 37 degree C for 2 hours.5. Heat inactivate the restriction digestion and then perform 1 % agarose gel run.6. Gel purify expected band of 692 bp using appropriate kit based methods.

Tips

Perform restriction digestion in 50 microliter volume with total 1 microgram of donor DNA. If you need to more DNA, then set up multiple reactions of 50 microliter volumes

Results Summary

Restriction of donor plasmid with BaeGI gave 2 bands of size 1549 bp and 692 bp as seen in 1% agarose gel analysis. The band of size 692 bp was then gel purified for further cloning experiments.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy to follow protocol and almost complete digestion without star activity can be achieved within 2 hours of digestion.

The Bad

None

The Bottom Line

Satisfied with using NEB's BaeGI enzyme for digesting and getting 692 bp DNA fragment from recipient plasmid. Highly recommend using this enzyme for cloning experiments with your DNA or plasmid constructs having BaeGI restriction site.

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