Competent E.coli: To buy or not to buy?

by Nick on September 20, 2007

Buying competent cells from commercial suppliers is convenient, provides a guarantee of quality and gives access to strains with a variety of in-built traits that assist with things like maintenance of plasmid integrity (more on these traits later). However, this can be an expensive business. Alternatively, competent cells of any strain, including the specially-constructed commercial strains, can be prepared easily in the lab (for protocols see here and here). The critical factor is whether you can generate cells of high enough competence yourself.

Competence is a measure of how efficiently the cells can be transformed and is defined as the number of colonies formed per Âµg of supercoiled DNA. It can be easily calculated transforming the cells with a known amount of supercoiled DNA then dividing the number of colonies formed by the amount of DNA (in Âµg) used. The level of competency that is required depends on the application. Here are a few examples:

Competence (cfu/Âµg)	Application
>1×10⁹	Genomic library construction, Quikchange
1×10⁶	Routine cloning/sub-cloning
1×10³	Transformation of circular plasmid DNA

If you can generate cells with these sorts of competencies, then there is no need to buy in competent cells, unless you are from a super-rich lab, or use competent cells very infrequently. However, although the protocols are simple, obtaining home made cells with a high competence is a bit of a knack. Here are some tips that should help:

Start with a nice, freshly-streaked colony
Grow the cells in detergent-free glassware (wash well with DI water prior to autoclaving)
Harvest the cells at around OD=0.5 to 0.7 as this is when they are happiest
Put the cells on ice when they reach the required OD and keep them cold all through the protocol
Chill EVERYTHING before performing the protocol. This includes pipettes, tips, solutions, centrifuges etc. If you can do the whole thing in a cold room, even better! (A bit uncomfortable, but worth it!!)
Treat the cells very gently – keep pipetting to a minimum and centrifuge cells at low speed so that tight pellets are not formed. It is worth accepting that you will loose even as much as 50% of the cells throughout the procedure in exchange for gentle centrifugation and pipetting.
Work quickly, but carefully
For very high competencies, a simple trick is to resuspend the cells at a higher density in the final step – so there are less aliquots but more cells/aliquot.

Articles in this series:

Photo: Jessica Shannon

About the Author

Nick Oswald

Nick is a molecular biologist-turned-publisher. After a PhD in Developmental Biology and an eclectic seven years in biotech he is now Editorial Manager of Neuroendocrinology and the founder and Editor-In-Chief of Bitesize Bio. You are welcome to connect with Nick on LinkedIn

Discussions on this article

2 Responses to “Competent E.coli: To buy or not to buy?”

E.coli Electroporation vs Chemical Transformation | The Molecular Biology Blog says:

September 20, 2007 at 2:42 pm

[...] Competent E.coli: To Buy or Not to Buy? [...]
Choosing a Competent E.coli Strain | The Molecular Biology Blog says:

September 24, 2007 at 10:56 am

[...] This is the third of four articles on E. coli competent cells and transformation. Part 1, Part 2 [...]