Difference between revisions of "Team:Aix-Marseille/Experiments/Protocols"

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===3- Preparation of Competent Bacterial cells===
 +
* Transfer the Bacterial culture in 50 ml Falcon tubes
 +
* Centrifuge for 10 minutes at 3500 rpm in cold
 +
* Remove the supernatant then re-suspend the pellet in 20 ml of Tbf1 for 50 ml of bacteria
 +
* Centrifuge for 5 minutes at 3500 rpm in cold
 +
* Poll in all bacterial culture in a single tube
 +
* Remove the supernatant then re-suspend the pellet in 8 ml of Tbf2 for 200 ml of bacteria
 +
* Allocate 220 µl of competent Bacterial cells in each eppendorf tube
 +
* instant freeze the eppendorf tubes in liquid nitrogen
 +
* Conserve the tubes at -80˚C
 +
 +
NOTE: all handling done in a cold room. To re-suspend the pellet a hard jerking action
 +
applied on the tube. Alternatively, put on wheel for 10 minute.

Revision as of 12:48, 9 June 2017

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Protocols

Preparation of competent Bacterial cells

1- Preparation of the Bacterial culture

  • Recuperate the overnight bacterial culture
  • Determine the OD600 in a 1 ml Tank Spectro, dilute a 100µl of culture in 900µl of distilled LB
  • In 1L Erlenmeyer add 200 ml of LB and an appropriate volume of culture to have an OD600 = 0.1
  • Put the Erlenmeyer in an incubator for about an hour at 37˚C.
  • test the OD600 for the new culture
  • the OD600 should be between 0.4 and 0.6

All the handling done in a 15cm radius of an open flame for optimal sterility

2- Preparation of Buffer Tbf1 and Tbf2

Tbf1 buffer Total volume 80mL
KAc 1M 2.4 mL
MnCl2 0.5M 8 mL
KCl 1M 8 mL
CaCl2 0.1M 8 mL
GlY 80% 15mL
H2O 38.6 mL

3- Preparation of Competent Bacterial cells

  • Transfer the Bacterial culture in 50 ml Falcon tubes
  • Centrifuge for 10 minutes at 3500 rpm in cold
  • Remove the supernatant then re-suspend the pellet in 20 ml of Tbf1 for 50 ml of bacteria
  • Centrifuge for 5 minutes at 3500 rpm in cold
  • Poll in all bacterial culture in a single tube
  • Remove the supernatant then re-suspend the pellet in 8 ml of Tbf2 for 200 ml of bacteria
  • Allocate 220 µl of competent Bacterial cells in each eppendorf tube
  • instant freeze the eppendorf tubes in liquid nitrogen
  • Conserve the tubes at -80˚C

NOTE: all handling done in a cold room. To re-suspend the pellet a hard jerking action applied on the tube. Alternatively, put on wheel for 10 minute.