Team:Paris Saclay/Protocols/Transformation of competent e.coli by CaCl2

From 2014.igem.org

Revision as of 13:38, 12 August 2014

Cells preparation

Day 1 Made a preculture (5ml LB)

Day 2 Wtih 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 on 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 - freez at -80°C

Transformation

Ad xµl of plasmide in 100µl of competents cells

shake smoothly

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

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