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

(→‎Protocols)
Line 19: Line 19:
 
For 200 mL of culture:
 
For 200 mL of culture:
  
−
Preparation of 80 mL of Tbf1 Buffer:
+
===Preparation of 80 mL of Tbf1 Buffer===
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
Line 41: Line 41:
 
|}
 
|}
  
−
Preparation of 8 mL of Tbf2 Buffer:
+
===Preparation of 8 mL of Tbf2 Buffer===
 
{| class="wikitable"
 
{| class="wikitable"
 
|NaMOPS 0.2M
 
|NaMOPS 0.2M
Line 61: Line 61:
 
|500 µL
 
|500 µL
 
|}
 
|}
 +
 +
== Protocol #2 : Transformations ==
 +
===Plasmids transformation ===
 +
#Add 20 ng { } of plasmid to 100 μL { } of competent cells thawed in ice
 +
##Incubate 30-45 min in ice
 +
#Thermal shock : put tubes in the Thermomixer at 42°C for 2 min
 +
#Incubate 5 min in ice
 +
#Add 900 μL { } of LB
 +
#Incubate 1 hour at 37°C with agitation
 +
#Spread 100 μL { } on LB limp (with antibiotic)
 +
 +
 +
===Ligation transformation===
 +
#Add 20 ng of ligation product to 100 μL of competent cells thawed in ice
 +
#Incubate 30-45 min in ice
 +
#Thermal shock : put tubes in the Thermomixer at 42°C during 2 min
 +
#Incubate 5 min in ice
 +
#Add 900 μL of LB
 +
#Incubate 1 hour at 37°C with agitation
 +
#Centrifuge 5 min at 5000 rpm
 +
#Eliminate 850 μL { } of medium
 +
#Spread 150 μL on LB limp (with antibiotic)
 +
 +
To make negative control, follow the same procedure but without adding plasmids and spreading 300 μL

Revision as of 07:40, 13 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

Protocol #2 : Transformations

Plasmids transformation

  1. Add 20 ng { } of plasmid to 100 μL { } of competent cells thawed in ice
    1. Incubate 30-45 min in ice
  2. Thermal shock : put tubes in the Thermomixer at 42°C for 2 min
  3. Incubate 5 min in ice
  4. Add 900 μL { } of LB
  5. Incubate 1 hour at 37°C with agitation
  6. Spread 100 μL { } on LB limp (with antibiotic)


Ligation transformation

  1. Add 20 ng of ligation product to 100 μL of competent cells thawed in ice
  2. Incubate 30-45 min in ice
  3. Thermal shock : put tubes in the Thermomixer at 42°C during 2 min
  4. Incubate 5 min in ice
  5. Add 900 μL of LB
  6. Incubate 1 hour at 37°C with agitation
  7. Centrifuge 5 min at 5000 rpm
  8. Eliminate 850 μL { } of medium
  9. Spread 150 μL on LB limp (with antibiotic)

To make negative control, follow the same procedure but without adding plasmids and spreading 300 μL