TSS Competent E. coli Preparation
Updated 6/15/2015 06:58 PM
Introduction
Materials
- Equipment
- 250mL Erlenmeyer flask
- Centrifuge
- Incubator with rotation
- Spectrophotometer
- Disposables
- 51x 1.5mL PP tubes (snap-cap)
- 2x 50mL PP centrifuge tubes
Procedure
TSS Buffer Composition
Table2
A A | B B | |
1 1 | Final Volume (ml)* | 20 |
Table1
A A | B B | C C | D D | |
1 1 | Component | Stock (M) | Amount | Unit |
2 2 | MgCl2 | 2 | 0.300 | ml |
3 3 | DMSO | - | 1 | ml |
4 4 | PEG (3350 or 8000) | - | 2 | g |
5 5 | LB medium | to final volume | to final volume | - |
Protocol
- Inoculate 1mL of liquid medium (LB or SOC) with E. coli strain of choice in a 1.5mL PP tube (snap-cap) and culture overnight at 37°C with rotation.
- Prepare 100mL of liquid medium (LB or SOC) in a sterile 250mL Erlenmeyer flask.
- Seed the 1mL culture into the 250mL flask and incubate at 37°C with 225 rpm rotation.
-Use this time to prepare the TSS buffer
- Cultivate until the optical density of the culture under 600nm wavelength is 0.3 to 0.4 (0.3 ≤ OD600 ≤ 0.4) (should take approximately 2-3h).
-TSS buffer should be chilled by this point.
- Once the proper optical density has been achieved, transfer the 100mL of culture into 2x 50mL PP centrifuge tubes and centrifuge under 2,700xg for 10 min at 4°C.
- Resuspend each tube in 5mL of pre-chilled TSS buffer with gentle vortexing (≤50%).
- Chill TSS suspended cells on ice for 15 min. Prepare/label 50x 1.5mL PP tubes (snap-cap) during this time.
- Distribute 200 μL of TSS suspended cells to each 1.5mL PP tube while ensuring the cells remain well mixed.
NOTE: 100 μL will be used per transformation.
- Cells can be used immediately, or stored at -80°C.
Comments
GOOD！
This method of competent cell preparation follows the TSS preparation described by Chung & Miller in the citation below.
The method described in the publication is reported to have successfully prepared the follwing E. coli strains for transformation:
D1210, DH1, DH5alpha, GM161, HB 101, JA221, JC7623, JE5513, JM83, JM105, JM107, JM109, K 802, LE392, LK111, MC1061, MC4100, MJM413, MM294, MV1190, N 4830-1, RA2021, RB79li, RR1, RZ1032, SCS-1, SG1117, SG21166, SMH50, TG1, TP610A, TP2010, TP2339, UT5600, VJS697, XL-1
Additionally, we have successfully prepared BLR(DE3) for transformation using the SOC medium preparation. Once prepared, these cells (stored at -80°C) retained their competency 6 months later. Even after one year of storage, cells were found to retain competency; however, potential loss of efficiency was not analyzed.
See: TSS Competent E. coli Transformation Chung, C. T. & Miller, R. H. (1993). [43] Preparation and storage of competent Escherichia coli cells. In Methods Enzymol, pp. 621-627.