Preparation of TSS Competent Cells
Updated 10/24/2019 11:17 PM
Introduction
Materials
- Equipment
- Shaking Incubator
- Bacterial Culture tubes
- Refrigerated centrifuge that can fit 2mL microcentrifuge tubes and can pellet cells
- Chilled 1.5mL sterile microcentrifuge tubes
- Micropipettes and chilled pipette tips
- Dry ice or regular ice
- Ice box or cooler
- NanoDrop spectrophotometer
- Reagents
- LB Media (warm)
- Chilled TSS solution (sterile)
- E. coli cells
Procedure
Important information to be documented throughout this procedure
A A | B B | |
1 1 | OD value of each sample | |
2 2 | Exact time duration that the back-dilution was incubated | |
3 3 | Number of competent cell samples made | |
4 4 | Any alterations to the protocol | |
5 5 | Any notable observations and issues | |
6 6 | Date of experiment |
Important Notes:
- This protocol requires two days to perform. Plan accordingly!
- This protocol requires as cold of an environment as possible. Work in a cold environment.
Chill any equipment and labware that will be used in the experiment beforehand.
- Work quickly to avoid warming up any part of the procedure after cells are harvested. An increase in temperature during the protocol results in dramatically less efficiency.
- Make sure there is enough LB Media to have enough to use as a blank for the NanoDrop spectrophotometer.
- Treat the cuvettes gently. You do not want to get any fingerprints or marks on the sides of the cuvette, which are used to read the OD.
Procedure:
Day 1
- Create 2mL overnight E. coli cultures. Each 2mL culture will result in one sample (1.5mL tube) of competent cells to use in a transformation.
- Incubate cultures at 250rpm and 37C for 15-19 hours.
- Chill in the 4C fridge:
Working TSS solution
microcentrifuge tubes
p1000 pipette tips
Day 2
- CRITICALBack-dilute: After pipetting up and down to agitate the culture, transfer 30µL of overnight culture to pre-warmed 3 mL LB Media. (1:100 dilution)
The LB should be around 37C.
- Incubate culture for 1.5-2 hours at 37C and 250rpm.
CRITICALCHECK OD600 AT 0.5 HRS. THE CULTURES COULD OVERGROW BY 1.5-2 HRS.
- Enter “Measure Cell Cultures” screen on NanoDrop with cuvette option selected.
- Place a thin cuvette into the cuvette holder, with the long end perpendicular to the vector of measurement.
- Fill cuvette with 1.5mL of LB, then close lid.
- Press “blank” button on screen.
This uses LB media as a blank solution.
- Remove cuvette and pipette out 750µL of LB, then pipette 750µL of culture into cuvette.
- Take the rest of the culture and put on ice to stop the growth.
- Mix slightly with pipette.
- Place the cuvette into the cuvette reader.
- Press “measure” to obtain the OD600 value. Double this value to get the actual OD600 of the measured culture.
CRITICAL Do everything from here on down in a cold environment-- as cold as possible!
- From the culture tube, transfer 1mL culture to microcentrifuge tubes.
- Spin cells at 6500rpm for 5 minutes at 4C. A small pellet should form. You may spin it for more time if the pellet does not form or you think it should be pelleted more.
- Discard supernatant and resuspend the pellet in 100µL of ICE-COLD TSS solution
- Use immediately or store in -80C ultracold freezer.
Storage + Cleanup
- If cells will not be used, store in -80C ultracold freezer.
Highly recommended to flash-freeze cells in dry ice if possible before storage.
- Bleach the supernatant before disposal.
This protocol describes the preparation of competent cells for TSS chemical transformation. This special chemical transformation preparation method requires less steps and can be used to make cells competent as well as directly store in a -80 ultracold freezer.
TSS Chemical Transformation should be performed after this protocol.