IGEM:Melbourne/2008/Protocols/competent cells

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 9: Line 9:
==Procedure==
==Procedure==
-
1) Grow 130ml LB culture from the 5ml grew previously to
+
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

Revision as of 19:16, 21 April 2008

Return to Melbourne Homepage

Return to Protocols


  • 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

Personal tools