Competent cell preparation

Wednesday, October 08, 2003

Description
For preparation of competent bacterial cells (14), a glycerol cell culture stock of the respective E. coli strain is thawed and added to 50 ml of liquid media. This culture then is preincubated at 37degC for 1 hour, transferred to an incubator-shaker, and is incubated further for 2-3 hours. The cells are pelleted by centrifugation, resuspended in calcium chloride solution, and incubated in an ice-water bath. After another centrifugation step, the resulting cell pellet again is resuspended in calcium chloride to yield the final competent cell suspension. Competent cells are stored at 4degC, for up to several days.

Primary Author
Bruce A. Roe ( broe@ou.edu )

Affiliation
University of Oklahoma , United States

Co-Author(s)
Judy S. Crabtree
Akbar S. Khan

Procedure
1. Thaw a frozen glycerol stock of the appropriate strain of E. coli, add it to an Erlenmeyer flask containing 50 ml of pre-warmed 2xTY (1) media, and pre-incubate in a 37degC water bath for 1 hour with no shaking. Further incubate for 2-3 hours at 37degC with shaking at 250 rpm.

2. Transfer 40 ml of the cells to a sterile 50 ml polypropylene centrifuge tube, and collect the cells by centrifugation at 3000 rpm for 8 minutes at 4deg C in a GPR centrifuge (Beckman) or 6000 rpm for 8 minutes at 4degC in an RC5-B centrifuge (DuPont) equipped with an SS-34 rotor. For M13-based transformation, save the remaining 10 ml of culture in an ice-water bath for later use.

3. After centrifugation, decant the supernatant and resuspend the cell pellet in one-half volume (20 ml) of cold, sterile 50 mM calcium chloride, incubate in an ice-water bath for 20 minutes, and centrifuge as before.

4. Decant the supernatant and gently resuspend the cell pellet in one-tenth volume (4 ml) of cold, sterile 50 mM calcium chloride to yield the final competent cell suspension.

Preparation of competant cells for frozen stroage

1. Innoculate 50 mls of 2xTY with a 2 ml overnight culture of GM272 and incubate in the shaker at 37degC for 2 to 2.5 hours.

2. Chill cells on ice for 10-15 minutes and transfer to a 50 ml polypropylene centrifuge tube and spin at 6 krpm for 5 minutes at 4degC.

3. Resuspend cell pellet in 20 ml of Frozen Storage Buffer (FSB by gentle vortexing and incubate on ice for 10-15 minutes.

4. Centrifuge as before and resuspend cell pellet in 4 ml FSB. Place on ice.

5. Add 140 ul of fresh DMSO and incubate on ice for 15 minutes. Add an additional 140 ul DMSO and keep on ice for 5 more minutes.

6. Transfer cells to sterile Falcon culture tubes (210 ul per tube). At this point in the procedure, the compentant cells may be placed at -70degC and stored indefinately.

7. To use compentant cells for transformation, remove from freezer and thaw for a few minutes at 37degC. Place on ice, add plasmid DNA and incubate for one hour as in the standard transformation procedure. Then heat shock at 42degC for 2 minutes, cool briefly, add 1 ml of 2xTY and incubate for 1 hour at 37degC before spreading on plates.

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Competent cell preparation