Team:Paris Saclay/Protocols/Transformation of competent e.coli by CaCl2
From 2014.igem.org
(Created page with "J1") |
|||
Line 1: | Line 1: | ||
- | + | ===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