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Revision as of 13:13, 12 August 2014 (view source) MelaCriq (Talk | contribs) (Created page with "J1") ← Older edit		Revision as of 13:37, 12 August 2014 (view source) MelaCriq (Talk | contribs) Newer edit →
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-	J1	+	===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

---

Revision as of 13:37, 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