Revision as of 09:52, 25 August 2014

Transformation of competent E. coli by CaCl₂

Day 1 Make a preculture (5ml LB)

Day 2 With precultures made the day before, add 1ml of bacteria in 100ml of LB medium - use a 500ml erlenmeyer

Shake vigorously at 37°C utile OD650 reaches 0.2/0.3

Cool down the culture by immersing them into ice

Centrifuge the cells for 10min at 4000 rpm at 4°C

Resuspend the cell pellet in 50 ml of CaCl₂ (initial volume/2)

5 min in ice

Centrifuge the cells for 10min at 4000 rpm at 4°C

incubate 3/4h in ice

cells stay competent during 24h at 4°C (increase of competence before a couple of hours in ice)

conservation = ad 1ml of glycerol 87% in 4ml of bacteria - freeze at -80°C

Add xµl of plasmid in 100µl of competent cells

shake smoothly

let 20min at 4°C 2min30s at 42°C (heat shock)

1-2min in ice

ad 0.9ml of LB medium without antibiotics

let 30min/1h to allow the expression of gene resistance

Spread on dishes (LB+Antibiotics) -100µl -200µL

centrifugate the rest of the sample - resuspend the cell pellet in 100µl of LB medium and spread on a dish

@@ Line 25: / Line 25: @@
 incubate 3/4h in ice
-cells stay competent during 24h at 4)C (increase of competence before a couple of hours in ice)
+cells stay competent during 24h at 4°C (increase of competence before a couple of hours in ice)
-conservation = ad 1ml of glycerol 87% in 4ml of bacteria - freez at -80°C
+conservation = ad 1ml of glycerol 87% in 4ml of bacteria - freeze at -80°C
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