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

(→‎2- Preparation of Buffer Tbf1 and Tbf2)
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|500 µL
 
|500 µL
 
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Some solution aren’t available directly and are prepared from the crystal form
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{|
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|
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|Volume (mL)
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|Mass (g)
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|Molecular weight (g/mol)
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|MnCl{{ind|2}} 0.5 M
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|200
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|19.791
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|197.91
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|KCl 1 M
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|200
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|17.91
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|74.55
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|NaMOPS 0.2 M
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|50
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|2.0926
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|209.26
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|KAc 1M
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|50
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|4.9
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|98.15
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Sterilize the flask prepared after weighing in an autoclave. When preparing Tbf1 and Tbf2 should be in proximity of an open flame
  
 
===3- Preparation of Competent Bacterial cells===
 
===3- Preparation of Competent Bacterial cells===

Revision as of 14:05, 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
Tbf2 buffer Total volume 8 mL
NaMOPS 0.2 M 400 µL
CaCl2 0.1 M 6 mL
Gly 80% 1.5 mL
KCl 1 M 80 µL
H2O 500 µL

Some solution aren’t available directly and are prepared from the crystal form

Volume (mL) Mass (g) Molecular weight (g/mol)
MnCl2 0.5 M 200 19.791 197.91
KCl 1 M 200 17.91 74.55
NaMOPS 0.2 M 50 2.0926 209.26
KAc 1M 50 4.9 98.15

Sterilize the flask prepared after weighing in an autoclave. When preparing Tbf1 and Tbf2 should be in proximity of an open flame

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.