Electroporation of A. tumfaciens cells

Monday, November 10, 2003

Description
Electroporation of A. tum faciens cells

Procedure
1. Make a overnight culture of the A.tum. strain at 28 C.
2. Dilute cefis 10 times in the desired medium (8 ml for 1 etectroporation).
3. Grow cells at 28 C until OD600 is between 0.5 -1.0
4. Spin at 4000 g for 10 min. at 4oC
5. Wash cells in Millipore water, twice the original volume (cold).
6. Spin again.
7. Wash ceils in 10% giycerol, the original volume (cold).
8. Spin again.
9. Resuspend ceils in a 200 fold smaller volume of 10% giycerof.
10. 40 ul of this suspension is used per eiectropcration.
11. Ceils can be stored at -80 C for about a month.
12. 1 ul of miniprep DNA is more than sufficient.
13. Eiectroporate at 2.5 kV, 200 W and 25 uF. The time constant should be between 4.6 and 4.9.
14. After efectroporation add 960 ul SOC and put the cells at 28 C for 1 hour.
15. Plate your cells in several dilutions.

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Electroporation of A. tumfaciens cells