TITLE: Preparation and transformation of chemically super-competent Escherichia coli
AUTHORS: Andreas Sagen

[DESCRIPTION]
Based on a method produced by Inoue, et al.

Inoue, Nojima, H., & Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids.Gene,96(1), 23–28. https://doi.org/10.1016/0378-1119(90)90336-P

[STEPS]
SECTION: Preparation of Inoue transformation buffer
1. In a sterile flask, add 200 mL distilled water
SECTION: Transformation
21. Quickly thaw a single reaction tube with 100 µL hyper-competent cells in Inoue-DMSO
SECTION: Preparation of Inoue transformation buffer
2. Measure 2.5 mL (1 Molarity (M)) PIPES, 2.177 g Manganese(II) chloride tetrahydrate, 3.75 mL (1 Molarity (M)) Calcium chloride and 4.660 g Potassium chloride.

Materials:
 
  
 
 
SECTION: Preparation of Inoue transformation buffer
6. Filter sterilize solution with a filter (0.2 µm) and store refrigerated (4 °C)
SECTION: Preparation of Inoue transformation buffer
5. Fill flask with distilled water to 250 mL
SECTION: Preparation of Inoue transformation buffer
4. Adjust pH to pH 6.7 with Potassium hydroxide solution

Materials:
 
SECTION: Preparation of Inoue transformation buffer
3. Add measured reagents and mix for 5 min
SECTION: Preparation of chemically super-competent cells
7. Prepare a culture of E. coli on an LB agar plate. Pick a single colony, and inoculate in 500 mL S. O. C. broth in a 1000 mL flask.

Incubate at 18 °C with shaking at 100 rpm overnight, until OD600 reaches 0.6
SECTION: Preparation of chemically super-competent cells
12. Incubate cells on ice for 10 min
SECTION: Preparation of chemically super-competent cells
11. Discard supernatant and resuspend with 16 mL CRM transformation buffer
SECTION: Preparation of chemically super-competent cells
10. Centrifuge tubes with 5000 rcf, 10 min, 4 °C
SECTION: Preparation of chemically super-competent cells
9. Place tubes on ice for 10 min
SECTION: Preparation of chemically super-competent cells
8. Aliquot entire culture volume into 50 mL canonical tubes
SECTION: Preparation of chemically super-competent cells
16. Discard supernatant and resuspend with 10 mL DMSO-Inoue per tube
SECTION: Preparation of chemically super-competent cells
15. Centrifuge tubes with 5000 rcf, 10 min, 4 °C. Meanwhile, prepare 50 mL DMSO-Inoue transformation buffer by diluting 3.5 mL DMSO in 46.5 mL Inoue transformation buffer

Materials:
 
SECTION: Preparation of chemically super-competent cells
13. Centrifuge tubes with 5000 rcf, 10 min, 4 °C
SECTION: Preparation of chemically super-competent cells
14. Discard supernatant and resuspend with 8 mL Inoue transformation buffer. Pool into two tubes
SECTION: Preparation of chemically super-competent cells
19. Snap freeze tubes in liquid nitrogen using a floating foam tube rack (Southern labware #HS2166)
SECTION: Preparation of chemically super-competent cells
18. Aliquot 100 µL cell suspension into sterile 500 µL screw cap reaction tubes (Sarstedt #72.704.200).

 

SECTION: Preparation of chemically super-competent cells
17. Incubate cells on ice for 30 min
SECTION: Preparation of chemically super-competent cells
20. Transfer aliquots storage box, and place in an ultra-low temperature freezer or vapor-phase nitrogen tank

 

SECTION: Transformation
23. Incubate cells on ice for 30 min
SECTION: Transformation
22. Mix 1-5 µL plasmid (ligation product)

 

 

SECTION: Transformation
24. Heat-shock cells at 42 °C for 30 s, followed by 2 min at 4 °C immediatly
SECTION: Transformation
25. Add 500 µL prewarmed S. O. C. medium and incubate for 37 °C at 200 rpm for 60 min
SECTION: Transformation
26. Add desired amount of suspension on LB plates with ampicillin (100 µg/mL) and incubate overnight