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 rpm
10. Resuspend the
cells in 200 ul fresh YENB and add 30 ul of 20 mg/ml XGAL and 30 ul of 25 mg/ml
IPTG
11. 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).