Talk:TOP10 chemically competent cells
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.
BC 11:07, 6 February 2007 (EST): What OD should the seed cultures be at the time glycerol is added?