Restriction Cloning Using SpeI-HF

May 08, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

SpeI-HF

Catalog Number:

R3133S

I have used SpeI-HF restriction enzyme from NEB to digest the plasmid called pSSPgl of a size 24 Kb. Digestion resulted in 23 and 1 Kb DNA fragments as seen in 0.7% agarose gel run. The 23 Kb DNA fragments (plasmid backbone) was gel excised and purified and then used for restriction cloning with similarly digested DNA insert of interest.

Experimental Design and Results Summary

Application

Restriction cloning of plasmids

Starting Material

1 microgram of plasmid DNA

Protocol Overview

1 microgram of pSSPgl5 plasmid DNA (prepped by Qiagen plasmid midi kit) was digested with 1 microliter of SpeI-HF in a 50 microliter reaction using Cutsmart buffer. Reaction mix was incubated at 37 degree C for 2 hr. Then digestion was stopped by adding 6X DNA loading purple dye. Digested mix was analysed by 0.7 % agarose gel and 23 Kb DNA fragment was gel excised and purified.

Tips

Larger plasmids should be preferably prepped by midikit method and should be of high quality with respect to purity.

Results Summary

Digestion of pSSPgl plasmid with SpeI-HF gave two DNA fragments of size 23 and 1 Kb. The 23 Kb DNA band (as seen in 0.7% agarose gel run) was gel excised and purified using NEB's Monarch DNA gel extraction kit method.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy to follow protocol and time saver qualified.

The Bad

None

The Bottom Line

Nearly complete digestion of the plasmid construct pSSPgl was achieved with SpeI-HF within 2 hours. Satisfied with using this enzyme for restriction cloning experiments with my plasmid construct.

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