Revision as of 07:41, 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

Preparation of 8 mL of Tbf2 Buffer

Protocol #2 : Transformations

Plasmids transformation

1. Add 20 ng { } of plasmid 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 for 2 min
4. Incubate 5 min in ice
5. Add 900 μL { } of LB
6. Incubate 1 hour at 37°C with agitation
7. 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