Jun 19, 2023
TSS competent cells and transformation
- 1Universidad Autónoma de Madrid
Protocol Citation: David Ruano Gallego 2023. TSS competent cells and transformation . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jzbrg2w/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: April 11, 2023
Last Modified: June 19, 2023
Protocol Integer ID: 80304
Abstract
This protocol allows the efficient transformation of plasmid into E. coli and related strains.
Materials
Transformation Storage Solution (TSS) buffer
- PEG3350 10%. Sigma. Ref: 202444
- DMSO 5%. DMSO (dimethyl sulfoxide)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8418
- Glycerol 10% Glycerol - for molecular biology, ≥99%Merck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
- MgCl2 20 mM. 1 M Magnesium Chloride (MgCl2)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M8266
- LB medium 2x.
Adjust to pH 6.1 with 6 M HCl.
Autoclave.
Add previously sterilized MnCl2 4 M to a final concentration of 140 mM.
Add water to adjust the modified LB medium to 1x.
Prepare aliquots of 1 ml and store at -20ºC.
5x KCM solution
- KCl 0.5 M. Potassium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9541
- Cacl2 150 mM. Calcium chloride dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #C7902
- MgCl2 250 mM. Magnesium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #M8266
Sterilize at 121ºC for 20 min. Store at 4ºC.
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.
Preparing competent cells
Preparing competent cells
2d
2d
Grow o/n a 5 mL culture of the strain to be transformed.
5 mL 37 °C Overnight
12h
Dilute 1:100 of the preinoculum in 50 mL prewarmed LB (OD600 = 0,05).
50 mL 37 °C
Grow at 37ºC with agitation (170 rpm) until it reaches OD600 = 0,5.
170 rpm, 37°C
1h 30m
Centrifuge the culture at 4000 g and 4ºC for 10 min.
4000 x g, 4°C, 00:10:00
10m
Discard supernatant and wash with 1 mL of chilled TSS.
1 mL On ice
Centrifuge at 4000 g and 4ºC for 3 min.
4000 x g, 4°C, 00:03:00
3m
Discard supernatant and resuspend in 1 mL of TSS.
1 mL On ice
Incubate 10 min on ice.
On ice 00:10:00
10m
Store 30 µL aliquots at -80ºC.
30 µL -80 °C
Transformation protocol
Transformation protocol
1h 40m
1h 40m
Mix 5 μL of 5x KCM solution with DNA (around 1-5 μL) and H2O to a total of a 25 μL mixture.25 µL
Mix the mixture with 25 μL of competent cells.
25 µL
Incubate 30 min on ice.
On ice 00:30:00
30m
Thermal shock: incubate at 42ºC for 90 s.
42 °C 00:01:30
1m 30s
Incubate 2 min on ice.
On ice 00:02:00
2m
Recovery: add 250 µl of LB and incubate at 37ºC for 1 h.
37 °C 01:00:00
1h
Plate 100 µl of the solution on LB agar plates supplemented with the indicated antibiotic and incubate o/n at 37ºC.
37 °C Overnight
12h