Electrocompetent E. coli for Transforming Large Constructs

September 12, 2018

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

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Company:

New England Biolabs

Product Name:

NEB 10 Beta Electrocompetent E.coli cells

Catalog Number:

C3020K

I have constructed large plasmid having gene interest to clone into recipient bacteria by Gibson assembly method. The constructed plasmid construct was about 15 kb in size and was successfully transformed into NEB 10 Beta Electrocompetent E. coli cells with high efficiency. Efficiency of transformation was 10^5 cells per 1 microgram of plasmid construct.

Experimental Design and Results Summary

Application

Transformation of large plasmid constructs for cloning purposes

Starting Material

2 microliter of 15 Kb plasmid construct per 50 microliter of NEB 10 beta electrocompetent cells

Protocol Overview

The protocol for electro-transformation was very simple. Just follow the easy to follow instructions given in the kit. Finally recover transformed cells in pre-warmed (at 37 degree C) SOC medium for 2 hr at 37 degree C shaking incubator. Spread appropriate dilutions onto selective agar plates and incubate them at 37 degree C. Next day pick some colonies and do colony PCR to check the presence of constructed plasmid in NEB 10 beta electrocompetent cells.

Tips

Thaw electrocompetent cells on ice for 15 minutes. Avoid vigorous shaking or pipetting of thawed cells.

Results Summary

Transformation efficiency with 15 Kb Gibson assembled plasmid construct in NEB 10 beta electrocompetent cells was 10^5 cells per microgram of the plasmid been transformed. Presence of the transformed plasmid construct was confirmed by colony PCR.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

High transformation efficiency and best choice for large plasmid constructs to be transformed with high efficiency.

The Bad

None

The Bottom Line

Ideal electrocompetent cells for difficult to transform and especially large plasmid cloning constructs of size above 10 Kb. Satisfied using these cells for my cloning plasmid constructs.

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