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

From 2014.igem.org

(Difference between revisions)
(Transformation of competent E. coli by CaCl2)
(Transformation of competent E.coli by CaCl2)
 
(2 intermediate revisions not shown)
Line 1: Line 1:
{{Team:Paris_Saclay/protocols_header}}
{{Team:Paris_Saclay/protocols_header}}
-
=Transformation of competent E. coli by CaCl2=
+
=Transformation of competent E.coli by CaCl2=
==Cell preparation==
==Cell preparation==
Line 28: Line 28:
conservation = ad 1ml of glycerol 87% in 4ml of bacteria - freeze at -80°C
conservation = ad 1ml of glycerol 87% in 4ml of bacteria - freeze at -80°C
-
 
-
{{Team:Paris_Saclay/protocols_footer}}
 
==Transformation==
==Transformation==

Latest revision as of 21:03, 17 October 2014

Transformation of competent E.coli by CaCl2

Cell preparation

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

Transformation

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