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

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

150_money.jpgBuying 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 µg 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 µg) used. The level of competency that is required depends on the application. Here are a few examples:

Competence (cfu/µg) 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

2 Comments

  1. Val on March 31, 2016 at 4:34 pm

    what mutations does the Top 10 DH10b E. coli have to use arabinose by araBAD promoter ie eGFParaBAD vector and where can this literature be found?

    • Ally on April 19, 2016 at 1:26 pm

      AraA and C have been deleted, AraD has the “139” allele which apparently makes it hypersensitive to repression by glucose. As a caveat, while browsing the genome on NCBI it seems to have two copies of AraD in different places, and I don’t know if they’re both the same allele or not.
      http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2293198/

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