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

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(Transformation of competent E.coli by CaCl2)
 
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===Cells preparation===
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{{Team:Paris_Saclay/protocols_header}}
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=Transformation of competent E.coli by CaCl2=
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==Cell preparation==
Day 1
Day 1
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Made a preculture (5ml LB)
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Make a preculture (5ml LB)
Day 2
Day 2
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Wtih precultures made the day before, add 1ml of bacteria in 100ml of LB medium - use a 500ml erlenmeyer
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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
Shake vigorously at 37°C utile OD650 reaches 0.2/0.3
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Centrifuge the cells for 10min at 4000 rpm at 4°C
Centrifuge the cells for 10min at 4000 rpm at 4°C
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Resuspend the cell pellet in 50 ml of CaCl2 (initial volume/2)
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Resuspend the cell pellet in 50 ml of CaCl<sub>2</sub> (initial volume/2)
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5 min on ice
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5 min in ice
Centrifuge the cells for 10min at 4000 rpm at 4°C
Centrifuge the cells for 10min at 4000 rpm at 4°C
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incubate 3/4h in ice
incubate 3/4h in ice
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cells stay competent during 24h at 4)C (increase of competence before a couple of hours in ice)
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cells stay competent during 24h at 4°C (increase of competence before a couple of hours in ice)
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conservation = ad 1ml of glycerol 87% in 4ml of bacteria - freez at -80°C
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conservation = ad 1ml of glycerol 87% in 4ml of bacteria - freeze at -80°C
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==Transformation==
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===Transformation===
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Add xµl of plasmid in 100µl of competent cells
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Ad xµl of plasmide (in 100µl of competents cells
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shake smoothly
shake smoothly
let 20min at 4°C
let 20min at 4°C
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2min30s at 42°C (heat choc)
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2min30s at 42°C (heat shock)
1-2min in ice
1-2min in ice
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-200µL
-200µL
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centrifugate the reste of the sample - resuspend the cell pellet in 100µl of LB medium and spread on a dish
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centrifugate the rest of the sample - resuspend the cell pellet in 100µl of LB medium and spread on a dish
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{{Team:Paris_Saclay/protocols_footer}}

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