Transformation Of Large Plasmid Constructs

April 02, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

NEB 10-beta Electrocompetent E.coli Cells

Catalog Number:

C3020K

I have designed and constructed large plasmid constructs for expression purposes. Plasmid construct was 23 Kb in size and needed high efficiency electrocompetent cells. Hence I used NEB 10-beta electrocompetent cells for this purpose and was satisfied with transformation efficiency. I got good number of colonies (around 300 colonies with 200 ng plasmid construct transformation). Colonies were screened by Colony PCR confirmed presence of plasmid in E.coli 10-beta cells (8 out 10 colonies gave correct colony PCR results).

Experimental Design and Results Summary

Application

Transformation of difficult and large plasmid constructs for cloning and expression

Starting Material

200 ng of 23 Kb plasmid in a 50 microliter of NEB-10 beta electrocompetent E.coli Cells

Protocol Overview

Electro-transformation protocol is very simple. Thaw the electrocompetent cells on ice for 20 to 30 minutes. Add 200 ng (ideally in less than 5 microliter volume) of plasmid construct. Transfer cells with plasmid into pre-chilled BioRad Gene pulser electroporation cuvette (0.2 cm gap). Pulse in BioRad Xcell gene pulser at 2500 volts current. Then immediately recover the cells in 37 degree C pre-warmed SOC medium for 2 hrs at 37 degree C with rotation of 180 rpm. Then spread 100 microliter of this transformation mix onto selective agar plate. Next day , observe the colony growth and screen positive colonies with colony PCR.

Tips

Make sure that electrocompetent cells been thawed on ice. Wipe out wet surfaces of electroporation cuvette to avoid arcing.

Results Summary

Satisfactory transformation of my 23 Kb plasmid construct using NEB 10-beta electrocompetent cells was observed. Got 300 colonies after spread plating of 100 microliter of transformation mix (200 ng plasmid construct) onto selective agar plate.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Efficient electrocompetent cells for large plasmid transformation. It comes with SOC medium, which is a plus.

The Bad

None

The Bottom Line

Overall satisfied with using NEB 10-beta electrocompetent E. coli cells for transforming my large plasmid constructs.

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