重组DNA的分离、克隆与测序实验手册-6
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).
F. Calcium Chloride treated bacterial cell transformation
A brief background discussion of transformation and transfection can be found in the Appendix.
For DNA transformation (14,15), the entire DNA ligation reaction is added to an aliquot of competent cells, which is mixed gently, and incubated in an ice-water bath. This mixture then is heat-shocked briefly in a 42degC water bath for 2-5 minutes. At this point in the transformation, the method varied slightly depending on whether the cloning vector is M13-based or pUC-based.
For M13-based transformation (14), an aliquot of non-competent cells is added to the heat-shocked mixture, as is the lac operon inducer homologue, IPTG, and the b-galactosidase chromogenic substrate, x-gal. Melted top agar is added, and the transformation mixture then is poured onto the surface of an agar plate. After the top agar solidified, the plates are inverted and incubated overnight at 37degC.
For pUC-based transformation (15), an aliquot of liquid media is added to the heat-shocked mixture, which then is incubated in a 37degC water bath for 15-20 minutes. After recovery, the cell suspension is concentrated by centrifugation and then gently resuspended in a smaller volume of fresh liquid media. IPTG and x-gal are added to the cell mixture, which is spread onto the surface of an ampicillin-containing agar plate. After the cell mixture had diffused into the agar medium, the plates are inverted and incubated overnight at 37degC.
Protocol
1. Add the entire ligation reaction to a 12 X 75 Falcon tube containing 0.2-0.3 ml of competent cells, mix gently, and incubate in an ice-water bath for 40-60 minutes. (For retransformation of recombinant DNA, add approximately 10-100 ng of DNA directly to competent cells).
2. Heat shock the cells by incubation at 42degC for 2-5 minutes.
For M13-based transformation:
3a. Add the following reagents to the heat shocked transformation mixture:
Non-competent cells 0.2 ml IPTG (25 mg/ml H2O) 25 ul x-gal (20 ml/ml DMF) 25 ul lambda top agar 2.5 ml
4a. Mix by briefly vortexing, and then quickly pour onto the surface of a pre-warmed lambda agar plate.
5a. Allow 10-20 minutes for the agar to harden, and then invert and incubate overnight at 37degC.
For pUC-based transformation:
3b. Add the following reagents to the heat shocked transformation mixture, add 1 ml of fresh 2xTY and incubate in a 37degC water bath for 15-30 minutes.
4b. Collect the cells by centrifugation at 3000 rpm for 5 minutes, decant the supernatant, and gently resuspend in 0.2 ml of fresh 2xTY.
5b. Add 25 ul IPTG (25 mg/ml water) and 25 ul x-gal (20 mg/ml DMF), mix and pour onto the surface of a pre-warmed LB-Amp plate. Spread over the agar surface using a sterile bent glass rod or sterile inoculating loop.
6b. Allow 10-20 minutes for the liquid to diffuse into the agar, and then invert and incubate overnight at 37degC.
For pBR322, pAT153 or other non-lacZ containing vectors:
3b. Add 1 ml of fresh 2xTY to the cells and incubate for 15-30 minutes at 37 degC. Spread approximately 50 ul on L plates containing antibiotic using a sterile glass spreader. Incubate the plates overnight at 37degC.
G. Microcentrifuge Tube Transformation
Microcentrifuge transformations are recommended when a single plasmid is being retransformed or for qualitative transformation experiments. Shotgun cloning experiments should be transformed using the large scale transformation, since the objective is to efficiently obtain transformation of hundreds of distinct recombinant plasmids.
1. Inoculate 50 ml of fresh 2xTY media with 3 to 5 ml of a fresh overnight culture of a suitable host strain (GM272) and incubate for 2 to 3 hours at 37deg C.
2. Transfer 1 ml of the culture into a 1.5 ml tube and centrifuge for 5 min at room temperature. Use 1 tube of culture per DNA sample to be transformed.
3. Decant supernatant, and resuspend the cell pellet in 500 ul (1/2 volume) of sterile, cold 50 mM calcium chloride. Gently vortex if necessary.
4. Incubate 5 min. on ice.
5. Centrifuge as before, decant and resuspend the competent cell pellet in 100 ul (1/10 volume) of calcium chloride.
6. Transfer each 100 ul sample of competent cells to chilled 12 x 75 mm Falcon tubes which contain 3 to 5 ul of DNA sample (about 2 ng/ul to 20 ng/ul).
7. Incubate on ice for 15 minutes.
8. Heat shock the sample at 42degC for 5 minutes.
9. Add 1 ml of fresh 2xTY to each sample and recover the cells by incubating at 37degC for 15 min.
10. For lacZ containing vectors add 25 ul of 20 mg/ml IPTG (in water) and 25 ul of 24 mg/ml X-Gal (in DMF).
11. Add 2.5 ml of soft top agar to each sample, vortex and quickly pour onto the surface of a TYE-AMP agar plate. Allow at least 15-30 min. for the agar to solidify.
12. Invert the plates and incubate overnight at 37degC.
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