6 replies to this topic

[mahsa]

when it comes down to chemically competent bacterial cells, I am telling you, there is no method what so ever to compete MOPS. It rockes!
has any one tried it befor?

[phage434]

Protocol? Reference?

[mahsa]

well, you are going to need
1- MOPS 0.5 molar : (please see A1.18 sambrook, vol.3, lOx MOPS Electrophoresis Buffer) 20 ml
2- KCl 7.4 g
3- CaCl2.2H2O 7.5 g
4- glycerol 100 ml

a) dissolve the aforementioned constituent in 1 liter of milliQ water and autoclave. now you have got FB buffer
inoculate 25 ml of LB with 250 ul of refreshed bacteria
c) incubate at 37 centigrade degrees till its optical density at 600 nm reaches 0.4-0.5
d) incubate on ice for 60 min
e) centrifuge at 4 centigrade degrees/3000 rpm/15 min
f) discard the supernate and resuspend the bacterial pellet in FB buffer(1/3 of the starting volume)
g) incubate on ice for 60 min
h) centrifuge at  4 centigrade degrees/3000 rpm/15 min
i) discard the supernate and resuspend the bacterial pellet in FB buffer(1/12 of the starting volume)
j) aliquot the resulting chemically competent bacteria and keep it in minus 70 .

[Curtis]

MOPS and MES work the same way

[mahsa]

Yea, have heard about that, but never tried it before

[mahsa]

I wonder what is the mechanism  by which MOPS or MES make bacteria super competent? I would appreciate it if any one could come up with a reference

[bob1]

What sort of transformation efficiencies are you getting with the MOPS/MES systems?