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1 RbCl Competent Cell (E. coli) Preparation o 1.1 Introduction

o 1.2 Materials and Equipments

o 1.3 Protocol

o 1.4 Note

RbCl Competent Cell (E. coli) Preparation

Introduction

Escherichia coli cells are grown to log phase. Exposure to calcium ions renders the cells able to take up DNA, or competent. Cells are treated with a hypotonic solution containing RbCl expand. As a result, the expulsion of membrane proteins allows negatively charged DNA to bind. The mixture of DNA and cells is then heat shocked or electroporated, which allows the DNA to efficiently enter the cells.

Materials and Equipments

50 mL LB media in a 250 mL culture flask pre-cooled (4°C) RF1 and RF2 buffers

pre-cooled (-80°C) steriled microcentrifuge tubes

RF1 buffer

RbCl

MnCl2·4H2O

Potassium Acetate

CaCl2·2H2O

Glycerol

100mM (12g/L)

50mM (9.9g/L)

30mM (2.9g/L)

10mM (1.5g/L)

15%(w/v)

   Adjust pH to 5.8 with acetic acid. Sterilize. Store in light-proof environment.

RF2 buffer

RbCl

MOPS

CaCl2·2H2O

Glycerol

10mM (1.2g/L)

10mM (g/L)

75mM (11g/L)

15%(w/v)

   Adjust pH to 6.8 with NaOH. Sterilize. Store in light-proof environment.

Protocol

1. Prepare fresh overnight 2 mL LB (with antibiotics if already containing plasmids) culture from plate colonies or frozen stocks.

2. Inoculate fresh overnight culture to 50 mL LB media in 1:250 (200μL) or 1:500 (100μL) dilution.

3. Incubate at 37°C with agitation until the Optical Density (OD600) reaches 0.4-0.5 (about 3hr).

4. Transfer the culture into 50 mL centrifuge tube and chill the culture on ice for 10-15 min.

5. Pellet the cells by centrifugation at 1000×g for 12 min at 4°C. Drain the pelleted cells thoroughly by inverting the tubes, and use pipette to draw off recalcitrant drops of media.

6. Resuspend the cell pellet with 15 mL of pre-cooled RF1 buffer. Do it on ice and quickly.

7. Incubate the cell suspension on ice for 15 min. And pellet cells as in step 5.

8. Resuspend the cell pellet with 4 mL of pre-cooled RF2 buffer. Do it on ice and quickly.

9. Incubate the cell suspension on ice for 15 min. And quickly aliquot 50μL (or 100μL) of the cells into pre-cooled microcentrifuge tubes.

10. Immediately freeze the cells in a dry ice/isopropanol bath or liquid nitrogen, and store at -80°C.

Note

1. The growiing phase (O.D.600) of 50 mL culture is critically important to the quality of competent cells.

2. From the step 4 on, keep every process on ice and do it quickly. High temperature and long processing time will decrease the cell quality.

3. For saving time, ask somebody to help you opening, recapping and freezing the microcentrifuge tube while aliquoting the cell suspensions.

4. Test your competent cell efficiency (c.f.u.) with 1 pg of plamid DNA by conventional Heat-Shock transformation. The effeciency of a good-quality competent cell (For DH5α) should be higher than 107 c.f.u.

Topic Started by trook on 11/11/2006 19:34 PM   

Please find the link and protocol for the following method:

Competent cell preparationhttp://www.genome.ou.edu/protocol_book/protocol_partII.html#II.E

There are two main methods for preparation of competent bacterial cells (14) for transformation, the calcium chloride and the electroporation method. For the calcium chloride method, 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.

Calcium Chloride Protocol

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 calcium chloride competent cells for frozen storage

1. Transfer 166 ul of the competent cell suspension to sterile Falcon culture tubes. 2. Add 34 ul of sterile 100% glycerol to the 166 ul aliquots of the final competent cell suspension prepared above, giving a final concentration of 17 % glycerol. 3. The competent cells then should be placed at -70degC and can be stored indefinately. 4. To use competent 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.

Electroporation Protocol Preparation of Electro-competent Cells:

1. Grow XL1-Blue cells on a tetracycline plate (20 ug tet/ml of LB agar)2. Inoculate 3 ml of YENB and grow overnight at 37 degrees C with shaking at 250 rpm in the New Brunswick incubator shaker.3. Inoculate the 3 ml of overnight growth into 1 liter of YENB (7.5 grams of Bacto Yeast Extract and 8 grams of Bacto Nutrient Broth brought to 1 liter with distilled water and autoclaved) and grow to an A600 of 0.5 (typically requires 3-4 hours of shaking at 250 rpm in the New Brunswick incubator shaker at 37 degrees C.4. Distribute the 1 liter of cells into four 500 ml Sorval (GS-3) centrifuge bottles and centrifuge at 5000 rpm at 4 degrees C for 10 minutes.Note: Steps 5-9 should be performed in the cold room and typically ~600 ml of ice cold sterile water and 150 ml of ice cold sterile 10% glycerol are required for manipulating the cells from a 1 liter growth.5. Resuspend each pellet in 100 ml of ice cold sterile double distilled water and combine the resuspended pellets into two Sorval centrifuge bottles (i.e each bottle then will contain 200 ml of resuspended pellet).6. Centrifuge at 5000 rpm at 4 degrees C for 10 minutes in the Sorval GS-3 Rotor.7. Resuspend each of the two pellets in 100 ml of ice cold sterile double distilled water and combine the resuspended pellets into one Sorval centrifuge bottle and centrifuge at 5000 rpm at 4 degrees C for 10 minutes in the Sorval GS-3 Rotor once more. Note: The purpose of all these centrifugation/resuspension/centrifugation steps is to insure that the cells are essentially "salt-free" as salt causes arching during the electroporation step.8. Resuspend the pellet in 100 ml of 10% ice cold sterile glycerol, centrifuge as above, and finally resuspend the pellet in 2 ml of 10% ice cold sterile glycerol to give salt-free, concentrated electrocompetent cells.9. Aliquote 40 ul of these electrocompetent cells into small snap cap tubes and immediately freeze by placing in curshed dry ice and then store at -70 degrees C until needed.

Electroporation Protocol for transformations using double-stranded plasmids

1. Thaw the electro-competent cells on ice for about one minute.2. Add 2-3 ul of the ligation mix to the cells.3. transfer 40 ul of the cells into to BTX Electroporation cuvettes PLUS and MAKE SURE THAT THE CELLS COVER THE BOTTOM OF THE CUVETTE.4. Turn on the Bio Rad E. coli Pulser and set the current to 2.5 KV by pushing the "Lower" and "Raise" bottoms simultaneously twice.5. Place the cuvette in the holder and slide it into position.6. Charge by pressing the "Charge" bottom until you hear the beep.7. Immediately, suspend the cells in 1 ml of YENB and transfer into a Falcon tube.8. Incubate the cells at 37 degrees C for 30 minutes at 250 rpm shaker.9. Spin the cells in BECKMAN table-top centrifuge for 8 minutes at 2500 rpm10. Resuspend the cells in 200 ul fresh YENB and add 30 ul of 20 mg/ml XGAL and 30 ul of 25 mg/ml IPTG11. Plate ~130 ul of the cells on pre-warmed LB-amp plates.

Reference:

Rakesh C. Sharma and Robert T. Schimke, "Preparation of Electro-competent E. coli Using Salt-free Growth Medium", Biotechniques 20, 42-44 (1996).

5. Replies

omehenk United

Kingdom

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Posted By omehenk on 5/2/2007 11:39 AM   

A far better method for making chemically competent cells is theRbCl method (if done well, it can give you efficiencies >= 10^8)

T F B 1 , 200 mls: 0.59 g KOAc (30 mM)2.42 g RbCl (100 mM)0.29 g CaCl22H2O (10 mM)1.98 g MnCl24H2O (50 mM)160mg Hexamine cobalt Cl (3mM)30 ml Glycerol (15%)

Adjust to pH 5.8 with 0.2 M acetic acid (do not adjust pH with KOH).Add ddH2O to 200 ml. Filter sterilize. Store refrigerated at 4°C

T F B 2 , 200 mls: 0.42 g MOPS (10 mM)

2.21 g CaCl2-2H2O (75 mM)0.24 g RbCl (10 mM)30 ml Glycerol (15%)Adjust to pH 6.5 with KOH.Add ddH2O to 200 ml. Filter sterilize. Store refrigerated at 4°C

II. Preparation of Competent CellsPick a fresh colony or scrape some from a glycerol stock and grow up an overnight culture in LB, shake at 37C.Dilute overnight culture 1 in 100 into 200mL of LB (pre-warmed to 37C).Grow at 37C (shaken) for 2-3 h, until OD600 0.25-0.3.Incubate on ice for 5 min.Spin at 3000g, 5 min 4C. Discard supernatant.Resuspend pellet in 40 ml Tfb I for a 200 ml prep. Do NOT Vortex. Pipet gently.Incubate on ice, 5 min.Centrifuge at 3000g, 5 min 4C. Discard supernatant.Resuspend pellet in 4 ml Tfb II. Be extremely gentle. Do not pipet cells!Incubate on ice, 15 min.Make aliquots of 100 ul. Freeze on dry ice. Store at -80C.

6.

Tony Rook United States

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Posted By Tony Rook on 5/2/2007 14:46 PM   

omehenk:

Have you had experience with successfully preparing competent cell lines of gram positive spore formers?

If so, is the method you referred to appropriate for this application, or do you feel there is a better method to use?

Tony Rook