Talk:TOP10 chemically competent cells

From OpenWetWare
Revision as of 07:22, 23 August 2006 by Barry Canton (talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search

BC 11:22, 23 August 2006 (EDT): I used this protocol to make BL21(DE3) competent. I obtained these transformation efficiencies -

  • 1.50E6 when transforming .01ng of pUC18 DNA from Invitrogen
  • 2.25E5 when transforming 1.0ng of pUC18 DNA from Invitrogen

The second figure is more likely to be accurate as I got a very small number of colonies when transforming .01ng of DNA.

I deviated from the protocol in some ways that may or may not have reduced my transformation efficiencies.

  • BL21(DE3) grew up much faster than the TOP10 cells described in the protocol. After 14hrs, BL21(DE3) was at OD600 1.72 instead of OD600 0.5 after 16hrs as is suggested for TOP10. This meant I had to do a second dilution and allow the cells to regrow to OD 0.5
  • Time constraints meant that some of my incubations on ice were closer to 30mins than 20mins.
  • I incubated the transformed cells in 2XYT instead of SOC.
  • Time constraints meant that I only incubated the transformed cells for 30mins prior to plating.