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How to produce competent celss XL1 Blue w/o inserts - (Sep/30/2005 )

Greetings ,

I am curious how do you produce more competent cells of XL1 Blue or BL 21 cells. They are witout inserts an empty wector.What plasmids should i use to transform it?.I did it oncee but i forgot how ? i f orgot to write it down. Darn.Help mee pleassee .thnkss a lot m8's


Use electroporation.


DNA sequencing reagents

-Daniel Tillett-

i follow this simple protocol to make competent cells with very good efficiency, u can transform with any compatible plasmid
Competent cells preparation : calcium chloride method

1. subculture 300 ul overnight E. coli culture into 10 ml LB ( in 50 ml centrifuge tube), shaking, 37℃, 1-2 hrs
2. OD550 = 0.2-0.4
3. Centrifugation: 3000 rpm, 15 min; save pellet, decant the supernatant completely
4. Dissolve the pellet with 5ml ice/steriliezed 100mM CaCl2
5. Incubation on ice for 30 minutes
6. centrifugation: 4℃, 3000 rpm, 15 min; save pellet, discard supernatant
7. Resuspend the pellet with 1-2 ml ice/sterilized 100mM CaCl2
8. Incubation for 1 hr (or 12-16 hr, best) on ice
9. aliquot to eppendorf: 150 ul each, in 15% glycerol, store in -80℃ freezer

hope it helps