TSS competent cells and transformation
David Ruano Gallego
Abstract
This protocol allows the efficient transformation of plasmid into E. coli and related strains.
Before start
Prepare solution TSS and KCM.
When prepared, TSS has a cloudy appearance that disappears once autoclaved. Also, over time, TSS can seem to be contaminated without being so, since the polyethylene glycol tends to precipitate.
Steps
Preparing competent cells
Grow o/n a 5 mL culture of the strain to be transformed.
5mL 37°C
Dilute 1:100 of the preinoculum in 50 mL prewarmed LB (OD600= 0,05).
50mL 37°C
Grow at 37ºC with agitation (170 rpm) until it reaches OD600= 0,5.
170rpm
Centrifuge the culture at 4000 g and 4ºC for 10 min.
4000x g,4°C
Discard supernatant and wash with 1 mL of chilled TSS.
1mL On ice
Centrifuge at 4000 g and 4ºC for 3 min.
4000x g,4°C
Discard supernatant and resuspend in 1 mL of TSS.
1mL On ice
Incubate 10 min on ice.
4On ice 0h 10m 0s
Store 30 µL aliquots at -80ºC.
30µL -80°C
Transformation protocol
Mix 5 μL of 5x KCM solution with DNA (around 1-5 μL) and H2O to a total of a 25 μL mixture.25µL
Mix the mixture with 25 μL of competent cells.
25µL
Incubate 30 min on ice.
On ice``0h 30m 0s
Thermal shock: incubate at 42ºC for 90 s.
42°C 0h 1m 30s
Incubate 2 min on ice.
On ice 0h 2m 0s
Recovery: add 250 µl of LB and incubate at 37ºC for 1 h.
37°C 1h 0m 0s
Plate 100 µl of the solution on LB agar plates supplemented with the indicated antibiotic and incubate o/n at 37ºC.
37°C
