Chemically Competent cells by the TSS method

Based on: https://openwetware.org/wiki/TSS and https://openwetware.org/wiki/Preparing_chemically_competent_cells

Make the buffers fresh!

TSS Buffer

Check pH should be 6.5 (acidic)

Dissolve magnesium chloride in LB, add PEG 3350. Filter sterilze 0.2 um. Add sterilely DMSO

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Day 1

From a plate with correct selection markers, a preculture of cells were grown overnight at 37 with their appropriate antibiotics.

Day 2

Volume above of buffer are suitable for 500 mL of competent cells

1) Innoculate 500 mL of LB + antibiotics with 1:100 dilution of preculture. A good flask is 500 mL in 3L flask.

2) Grow until OD 0.4-0.6

3) Transfer cells to 50 mL falcon tubes (prechilled). Let sit on ice 10 minutes. Spin down cells in 50 mL falcon tubes (prechilled) at 3900g x 15 minutes.

4) Decant supernatant

5) Resuspend ALL tubes with a total of 25-50 mL of TSS (ie. this makes 5-10% of initial culture volume)

6) Aliquot 100 uL of cells per eppendorf. CHILL YOUR TUBES for best cells. Directly freeze in N2 then transfer to -80C.

Immediately test the cells and plate on antibiotic plates to make sure they have not picked up any resistance.

Immediately transform cells to determine their competency. Compare with cells from a previous batch

Previous times:

KL: 18.02.22 :  DH10EMBacY - 500 mL culture. 5 mL in 500mL of LB + Kan+Tet in 3L Flask. Started at 9:10. ready to be harvested 2H 45 minutes later. Resuspended in 25 mL, 100uL aliquots. TSS buffer we did not pH to 6.5 this time.