SEM (Inoue) competent cells - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

Competent E. coli cells according to the SEM protocol (Inoue et al. 1990) (Inoue) method. Cells prepared using this method can be used according to the Transforming Frozen Competent E. coli protocol. Some details are from this version. Detailed protocols can be found online

Needed glassware:

• One 1L Schott bottle
• One 1000 mL glass beaker
• One 1000 mL volumetric bottle
• One funnel
• One large (>2 cm) magnetic stirrer

Use clean glassware with no detergent residue. Clean glassware with 70% EtOH and let it dry. Glassware can also be autoclaved with some water to achieve the same result. It is best to set aside glassware for competent cell preparation that is not washed with detergent.

1. Prepare a solution (TB w/o Mn) in the 1000 mL beaker with the following components:

• 10 mM Hepes (or Pipes)
• 15 mM CaCl2
• 250 mM KCl

For 1 L (about ten preparations of about 80-100 tubes of competent cells each) this would mean:

• 2.39 g Hepes (238.3012 g/mol) or 3.03 g Pipes (302.37 g/mol)
• 1.67 g CaCl2 (110.98 g/mol) or 2.21 g CaCl2x2H2O (110.98 g/mol)
• 18.64 g KCl (74.5513 g/mol)

2. Dissolve the salts in ~ 800 mL water a large (1000 mL) glass beaker with a magnetic stirrer.
3. Adjust pH to 6.7 with KOH Potassium hydroxide (56.11 g/mol) 1M solution (‎3.3 g (85%) in 50 mL ).
4. Dissolve dry MnCl2 to have a final concentration of 55 mM. For 1 L this would mean 6.92 g MnCl2 (125.844 g/mol). MnCl2 is hygroscopic so it may stick to paper if used to weigh up the chemical. VERY IMPORTANT Add the MnCl₂ AFTER adjusting the pH or it will precipitate.

Sterilize the solution by filtration through 0.45 μm filter and store at +4°C. Alternatively, the solution can be made sterile by heating the solution in a microwave oven before adding all the water. Heat for 3-5 min at full power in a microwave oven with the cap shut tightly. Be careful when relieving the pressure. Add autoclaved water up to full volume. The buffer can be stored at 4°C indefinitely. Protocols do not usually recommend autoclaving, but according to this exchange, it should work as well.

1. Inoculate a 20-50 mL culture of E. coli in SB medium and let grow o/n at 37 C until saturated with vigorous (200-250 rpm) shaking.
2. Inoculate 200 ml of SOB medium in a 1-liter Erlenmeyer flask.
3. Grow to an OD640 = 0.6 at 18°C or at room temperature with vigorous shaking (200-250 rpm) or using a magnetic stirrer bar.
4. Cool the TB buffer on ice for several hours before use.
5. Divide cells into sterile 50 mL FALCON tubes and incubate on ice for 10 minutes to cool down.
6. Spin down cells at 2500 x g (i.e. 3000 rpm Beckman J-6B centrifuge or 3727 rpm in an Eppendorf 5804R) for 10 min at +4°C. The speed using the Eppendorf can be reduced at least as low as 3000 rpm without any problems. Spinning at lower speed makes it easier to resuspend the cells gently which allegedly improves competency.
7. Resuspend pellet in 60 ml of ice-cold TB (4 x 15 mL) and incubate on ice for 10 minutes, then spin down cells under the same conditions as above.
8. Resuspend pellet gently in 16 ml of TB.
9. Add DMSO to final concentration of 7%, Calculate the needed volume of DMSO like so:

V(DMSO) = 0.07*V(CELLS)/0.93;

V(CELLS) is the volume of cell suspension and V(DMSO) is the DMSO volume to add. Mix gently by swirling.

10. Incubate on ice for 10 minutes.
11. Dispense the cell suspension in 0.2 - 0.5 ml aliquots and immediately flash-freeze by immersion in liquid nitrogen.
12. Store frozen cells at -80°C