IGEM:Melbourne/2008/Protocols/competent cells

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Current revision (19:16, 21 April 2008) (view source)
 
Line 10: Line 10:
==Procedure==
==Procedure==
1) Grow 130ml LB culture from the 5ml grew previously to OD600 of NO MORE than 0.45
1) Grow 130ml LB culture from the 5ml grew previously to OD600 of NO MORE than 0.45
 +
2) Pour cultures into sterile 50ml falcon tubes
2) Pour cultures into sterile 50ml falcon tubes
 +
3) Pellet cells at 4 degree, 3000rpm for 5 min
3) Pellet cells at 4 degree, 3000rpm for 5 min
 +
4) Discard supernatant
4) Discard supernatant
 +
5) Resuspend pellet in total volume of 50ml ice-cold TFB1
5) Resuspend pellet in total volume of 50ml ice-cold TFB1
 +
6) Chill on ice for 5 min
6) Chill on ice for 5 min
 +
7) Pellet cells at 4 degree, 3000rpm for 5 min
7) Pellet cells at 4 degree, 3000rpm for 5 min
 +
8) Discard supernatant
8) Discard supernatant
 +
9) Resuspend pellet in 5 ml of ice-cold TFB2
9) Resuspend pellet in 5 ml of ice-cold TFB2
 +
10) Chill on ice for 15 min
10) Chill on ice for 15 min
 +
11) Aliquot on dry ice, then snap-freeze, then store at -80 degree
11) Aliquot on dry ice, then snap-freeze, then store at -80 degree

Current revision

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  • yield 5ml ~ 100 aliquots of 50µL
  • need another overnight culture (5ml)

Procedure

1) Grow 130ml LB culture from the 5ml grew previously to OD600 of NO MORE than 0.45

2) Pour cultures into sterile 50ml falcon tubes

3) Pellet cells at 4 degree, 3000rpm for 5 min

4) Discard supernatant

5) Resuspend pellet in total volume of 50ml ice-cold TFB1

6) Chill on ice for 5 min

7) Pellet cells at 4 degree, 3000rpm for 5 min

8) Discard supernatant

9) Resuspend pellet in 5 ml of ice-cold TFB2

10) Chill on ice for 15 min

11) Aliquot on dry ice, then snap-freeze, then store at -80 degree

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