Preparation of competent cells by CaCl2
Yishi Jin - 8/93
1. Inoculate 2-3ml SB with one colony of DH5a (recA-) or TG1, BB4 (recA+),
o/n culture at 37°C. NB sterile.
2. Dilute 0.25 ml of o/n culture into 25 ml SB in 125/250 ml flask. Add
0.25 ml M MgSO4 (or MgCl2) to get 10mM Mg++. Put in 37° shaker for
2-2.5 hr until OD600 is between 0.3 and 0.5.
3. Chill the culture for 5-10'. (Put CaCl2 on ice)
4. Spin down in 30 ml corex tube, 2500 rpm for 10'.
5. Pour off supernatant, resuspend pellet in 1/2 volume ice cold 50 mM
CaCl2.
6. Sit on ice for 15-30'.
7. Spin again 2500 rpm 10'. If the cells don't pellet nicely, try 3000
rpm spin 5'.
8. Pour off supernatant, resuspend pellet in 1/10 vol. CaCl2.
9. Sit on ice for 15-30'.
10. Cells now ready for transformation.
Imported from :
http://cajal.biology.utah.edu/JorgLabHomePage/
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