240
ANNETTE DEICHMANN AND KLAUS DEICHMANN
7. Centrifuge the cells at 4000 x g for 15 min at 4°C.
8. Resuspend the cells in 100 ml of ice-cold 10o/o glycerol solution.
9. Centrifuge the cells at 4000 x g for 15 min at 4°C.
10. Resuspend the cells in 50 ml of ice-cold glycerol solution.
11. Centrifuge the cells at 4000 x g for 15 min at 4°C.
12. Discard the supernatant and resuspend the cells in 1 ml of ice-cold 10o/o
glycerol solution.
13. The cells are now ready for use in electroporation. As well they can be
stored insmall aliquots at -70°C (before use gentlythaw the cells on ice).
Transformation 14. To an ice-cold Eppendorf tube add 40 Jll of the E. coli cell suspension
and 1-2 J.tl of DNA in a low ionic-strength buffer.
15. Mix well and incubate on ice for 1 min.
16. Set the Gene Pulser apparatus (Bio-Rad Laboratories) at 2.5 kV, 25J.1F
and 200 Ohms parallel resistance. The time constant achieved by the
above settings equals approximately 4.7 msec.
17. Transfer the mixture of cells and DNA to the bottarn of an ice-cold 0.2
cm electroporation cuvette.
18. Apply one pulse at the above settings. This should result in a time constant of approximately 4.5-5 msec.
19. Immediatelyafterthepulse, add 1 mlofSOCmedium to the cuvette and
mix gently.
20. Transfer the transformed cells to a culture tube and incubate at 37°C for
1 hr with vigoraus shaking.
21. Spread different amounts of the culture ( e.g. 50, 100, and 200 J.tl) over
the surface of LB agar plates containing the appropriate antibiotic. Incubate the plates inverted in an incubator at 37°C and let the cells grow
overnight.
22. Identify the recombinant clones of e.g. pBR322 as described in Subprotocoll.
ANNETTE DEICHMANN AND KLAUS DEICHMANN
7. Centrifuge the cells at 4000 x g for 15 min at 4°C.
8. Resuspend the cells in 100 ml of ice-cold 10o/o glycerol solution.
9. Centrifuge the cells at 4000 x g for 15 min at 4°C.
10. Resuspend the cells in 50 ml of ice-cold glycerol solution.
11. Centrifuge the cells at 4000 x g for 15 min at 4°C.
12. Discard the supernatant and resuspend the cells in 1 ml of ice-cold 10o/o
glycerol solution.
13. The cells are now ready for use in electroporation. As well they can be
stored insmall aliquots at -70°C (before use gentlythaw the cells on ice).
Transformation 14. To an ice-cold Eppendorf tube add 40 Jll of the E. coli cell suspension
and 1-2 J.tl of DNA in a low ionic-strength buffer.
15. Mix well and incubate on ice for 1 min.
16. Set the Gene Pulser apparatus (Bio-Rad Laboratories) at 2.5 kV, 25J.1F
and 200 Ohms parallel resistance. The time constant achieved by the
above settings equals approximately 4.7 msec.
17. Transfer the mixture of cells and DNA to the bottarn of an ice-cold 0.2
cm electroporation cuvette.
18. Apply one pulse at the above settings. This should result in a time constant of approximately 4.5-5 msec.
19. Immediatelyafterthepulse, add 1 mlofSOCmedium to the cuvette and
mix gently.
20. Transfer the transformed cells to a culture tube and incubate at 37°C for
1 hr with vigoraus shaking.
21. Spread different amounts of the culture ( e.g. 50, 100, and 200 J.tl) over
the surface of LB agar plates containing the appropriate antibiotic. Incubate the plates inverted in an incubator at 37°C and let the cells grow
overnight.
22. Identify the recombinant clones of e.g. pBR322 as described in Subprotocoll.