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Team:Paris Saclay/Protocols/Transformation of competent e.coli by CaCl2
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| + | =Transformation of competent E.coli by CaCl2= | ||
| + | |||
| + | ==Cell preparation== | ||
Day 1 | Day 1 | ||
| - | + | Make a preculture (5ml LB) | |
Day 2 | 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 | Shake vigorously at 37°C utile OD650 reaches 0.2/0.3 | ||
| Line 13: | Line 17: | ||
Centrifuge the cells for 10min at 4000 rpm at 4°C | Centrifuge the cells for 10min at 4000 rpm at 4°C | ||
| - | Resuspend the cell pellet in 50 ml of | + | Resuspend the cell pellet in 50 ml of CaCl<sub>2</sub> (initial volume/2) |
| - | 5 min | + | 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 | ||
| Line 21: | Line 25: | ||
incubate 3/4h in ice | incubate 3/4h in ice | ||
| - | cells stay competent during 24h at | + | 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 - | + | 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 | shake smoothly | ||
let 20min at 4°C | let 20min at 4°C | ||
| - | 2min30s at 42°C (heat | + | 2min30s at 42°C (heat shock) |
1-2min in ice | 1-2min in ice | ||
| Line 45: | Line 48: | ||
-200µL | -200µL | ||
| - | centrifugate the | + | centrifugate the rest of the sample - resuspend the cell pellet in 100µl of LB medium and spread on a dish |
| + | |||
| + | {{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
