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Latest revision as of 08:50, 10 September 2015

""

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competent cells (new)

e.g. protocol for DNA-isolation
remarks concerning the protocol (author, adapted from etc.)

material: chemicals, used kits, …
duration: … min

Protocol

  • streak out E. coli cell on LB plate without (or with appropriate antibiotics), grow over night at 37°C
  • inocculate 5 ml o/n culture with one colony
  • inocculate 100 ml LB with 1 ml o/n culture, grow for 2-3 h until an OD(600)=0,4 to 0,6
  • transfer bacteria into pre-cooled and sterile centrifuge tube (250 ml) and pellet bacteria 5 min (5000 rpm; 4°C)
  • discard supernatant

from now on: work in cold room

  • resuspend pellet in 20 ml cold TFB1 with cut tips
  • incubate 5 min on ice
  • centrifuge 5 min (5000 rpm; 4°C)
  • resuspend pellet in 4 ml cold TFB2 with cut tips
  • aliquot 50 ml in screw cap eppis and freeze in liquid nitrogen
  • store at -80°C

buffer preparation

TFB1200ml
30 mM KAc0,589 g
50 mM MnCl21,6919 g
100 mM KCl1,482 g
10 mM CaCl20,294 g
15 % Glycerol30 g

* sterile filtrate and store at 4°C

TFB2200 ml
10 mM MOPS0,419 g
10 mM KCl0,149 g
75 mM CaCl22,205 g
15 % Glycerol30 g

* autoclave and store at 4°C

,