Talk:TOP10 chemically competent cells

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(Transformation efficiencies)
Current revision (17:26, 17 February 2013) (view source)
 
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==Questions==
==Questions==
*'''[[User:Bcanton|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?
*'''[[User:Bcanton|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?
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Please do not modify this protocol without discussion on this talk page.

Current revision

Comments

  • 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 prepped pUC18 DNA (1 colony on plate, so not very accurate number)
    • 2.25E5 when transforming 1.0ng of prepped pUC18 DNA (15 colonies on plate)

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 I stopped the cells growing at an OD600 of 0.6.
  • 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.
  • Reshma 15:06, 5 June 2007 (EDT): Growth of the overnight 250mL culture to OD600nm of 0.3 from seed stocks took exactly 17 hours at 20°C.
  • Andy Lane 12:31, 25 November 2010 (EST): Doubling time for DH5a or XL-10 Gold @ 20˚C in SOB media seems to be approx 2h 30m to 3h in my hands. This may help in planning the timing of the protocol.

Transformation efficiencies

  • Reshma 11:36, 12 June 2007 (EDT): Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.
  • Andy Lane 16:33, 26 November 2010 (EST): I achieved competencies of 8E7 for DH5a cells and 5.5E7 for XL-10 Gold cells. DH5a were harvested at OD600 of 0.3; XL-10 Gold at 0.35. However, I lost many cells during the centrifugation steps and froze down at a lower density than recommended. The protocol states that a 1:5 dilution in SOC should have an OD600 of 1-1.5 at freezing; mine was only 0.55 - 0.6 for both strains.

Questions

  • BC 11:07, 6 February 2007 (EST): What OD should the seed cultures be at the time glycerol is added?

Please do not modify this protocol without discussion on this talk page.

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