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

(→‎Protocol #1 : Preparation of competent bacteria cells)
(→‎Protocols)
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#Incubate 15 min in ice.
 
#Incubate 15 min in ice.
 
#Aliquot 200µL of cell suspension in sterile eppendorf tubes and conserve it at -80°C.
 
#Aliquot 200µL of cell suspension in sterile eppendorf tubes and conserve it at -80°C.
 +
 +
== Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers ==
 +
 +
For 200 mL of culture:
 +
 +
Preparation of 80 mL of Tbf1 Buffer:
 +
{| class="wikitable"
 +
|-
 +
|KAc 1M
 +
|2.4 mL
 +
|-
 +
|MnCl2 0.5M
 +
|8 mL
 +
|-
 +
|KCl 1 M
 +
|8 mL
 +
|-
 +
|CaCl2 0.1M
 +
|8 mL
 +
|-
 +
|Gly 80%
 +
|15 mL
 +
|-
 +
|H2O
 +
|38.6 mL
 +
|}
 +
 +
Preparation of 8 mL of Tbf2 Buffer:
 +
{| class="wikitable"
 +
|NaMOPS 0.2M
 +
|400 µL
 +
|-
 +
|CaCl2 0.1M
 +
|6 mL
 +
|-
 +
|KCl 1 M
 +
|8 mL
 +
|-
 +
|Gly 80%
 +
|1.5 mL
 +
|-
 +
|KCl 1M
 +
|80 µL
 +
|-
 +
|H2O
 +
|500 µL
 +
|}

Revision as of 14:32, 10 June 2016

Protocols

Protocol #1 : Preparation of competent bacteria cells

  Everything should be done in sterile conditions and on ice (4°C).
  1. Grow a culture of bacteria in LB medium until it reaches OD600 = 0.5.
    • While it grows, prepare Tbf1 and Tbf2 buffers.
  2. Centrifuge cells for 10 minutes at 3500g at 4°C
  3. Resuspend the pellet slowly in 80mL of Tfb1 buffer.
  4. Centrifuge 5 min at 3500g at 4°C.
  5. Resuspend the pellet in 8 mL of Tbf2 buffer.
  6. Incubate 15 min in ice.
  7. Aliquot 200µL of cell suspension in sterile eppendorf tubes and conserve it at -80°C.

Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers

For 200 mL of culture:

Preparation of 80 mL of Tbf1 Buffer:

KAc 1M 2.4 mL
MnCl2 0.5M 8 mL
KCl 1 M 8 mL
CaCl2 0.1M 8 mL
Gly 80% 15 mL
H2O 38.6 mL

Preparation of 8 mL of Tbf2 Buffer:

NaMOPS 0.2M 400 µL
CaCl2 0.1M 6 mL
KCl 1 M 8 mL
Gly 80% 1.5 mL
KCl 1M 80 µL
H2O 500 µL