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

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<h1>Protocols</h1>
 
<h1>Protocols</h1>
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==Preparation of competent Bacterial cells==
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===1- Preparation of the Bacterial culture===
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* Recuperate the overnight bacterial culture
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* Determine the OD600 in a 1 ml Tank Spectro, dilute a 100µl of culture in 900µl of distilled LB
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* In 1L Erlenmeyer add 200 ml of LB and an appropriate volume of culture to have an OD600 = 0.1
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* Put the Erlenmeyer in an incubator for about an hour at 37˚C.
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* test the OD600 for the new culture
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* the OD600 should be between 0.4 and 0.6
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 +
All the handling done in a 15cm radius of an open flame for optimal sterility
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===2- Preparation of Buffer Tbf1 and Tbf2===
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{|
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|Tbf1 buffer
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|Total volume 80ml
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|-
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|KAc 1M
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|2.4 mL
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|-
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|MnCl2 0.5M
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|8 mL
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|-
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|KCl 1M
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|8 mL
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|-
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|CaCl{{ind|2}} 0.1M
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|8 mL
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|-
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|GlY 80%
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|15ml
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|-
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|H{{ind|2}}O
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|38.6 mL
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|}

Revision as of 12:07, 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