Transformation of E. coli by Electroporation
Using electroporation to transform Escherichia coli results in transformation efficiencies
greater than can be obtained using the best chemical methods. It is easy to obtain
transformation efficiencies >108 per milligram DNA and efficiencies of 1010 have been
reported (Dower et al., 1988). Electroporation can be used to transform many different
types of bacteria.
APPENDIX 3N
BASIC
PROTOCOL
NOTE: All reagents and equipment coming into contact with live cells must be sterile.
Materials
LB medium (UNIT 10.18)
Transformation competent E. coli (e.g., HB101 or XL1 blue, Stratagene)
Sterile H2O (filter sterilize, see recipe), 4°C
15% (v/v) glycerol, sterile, 4°C
5 pg to 0.5 µg/µl DNA for transformation
SOC medium (see recipe)
100 mg/ml isopropyl β-D-thiogalactopyranoside (IPTG) in H2O (optional)
15 mg/ml 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (Xgal) in
dimethylformamide (optional)
LB plates with appropriate antibiotic (see recipe)
50-ml conical tube, sterile
Shaking incubator, 37°C
Fernbach or 2-liter flask
250-ml centrifuge bottles
Sorval or similar centrifuge with GSA rotor to accommodate 250-ml bottles, 4°C
5-ml pipets
Microcentrifuge, 4°C
Dry ice/ethanol bath
Electroporation cuvette, sterile
Electroporation apparatus
Prepare the bacteria
1. Place 5 ml LB medium in a 50-ml tube. Inoculate with one colony of transformation
competent E. coli and incubate overnight at 37°C with vigorous shaking.
2. Add the 5-ml overnight culture to 500 ml LB medium in a Fernbach flask. Incubate
with vigorous shaking at 37°C until the OD600 is between 0.5 and 1.0.
3. Cool the bacteria on ice ≥15 min.
4. Pour into two sterile 250-ml centrifuge bottles, maintaining sterility. Centrifuge 5 to
10 min at 2000 × g (4000 rpm in GSA rotor), 4°C.
5. Decant supernatant; remove any residual liquid with a sterile Pasteur pipet.
In all wash steps, it is critical to keep the bacteria cold and to remove all the supernatant;
otherwise too many residual ions will remain and the bacteria will not function for
electroporation.
6. Resuspend pellet in 500 ml cold sterile water and centrifuge 15 min at 2000 × g, 4°C.
The water used to resuspend the pellet must be cold.
7. Remove supernatant and resuspend the bacteria in 250 ml of 4°C sterile water.
Centrifuge 15 min at 2000 × g, 4°C and remove the supernatant.
Contributed by Sherie L. Morrison
Current Protocols in Immunology (1997) A.3N.1-A.3N.4
Copyright © 1997 by John Wiley & Sons, Inc.
Common
Immunologic
Techniques
A.3N.1
Supplement 21
8. Resuspend pellet in 10 ml of cold 15% glycerol, and centrifuge 15 min at 1500 × g
(3000 rpm in a GSA rotor), 4°C.
9. Decant supernatant. Aspirate the pellet and any residual glycerol into a sterile 5-ml
pipet and transfer to a 1.5-ml microcentrifuge tube.
10. Microcentrifuge 5 min at maximum speed, 4°C. Remove supernatant with a Pasteur
pipet and discard.
11. Resuspend pellet in 1 to 1.5 ml of cold 15% glycerol.
This procedure yields 2 to 4 × 1010 bacteria per milliliter.
12. Distribute 50-µl aliquots of the bacteria into sterile 1.5-ml microcentrifuge tubes.
Quick-freeze in a dry ice/ethanol bath and use immediately or store at −70°C for later
use.
If care is taken to prevent warming, bacteria can be stored several months at −70°C.
Transform competent bacteria
13. Thaw a tube of frozen bacteria on ice. Place an electroporation cuvette on ice to cool.
14. Add 1 µl of 5 pg to 0.5 µg/µl DNA for transformation to the thawed bacteria. Include
positive (supercoiled DNA at a known concentration) and negative (buffer or distilled
water) controls.
This volume of DNA is usually optimal. The danger is adding too much salt at this step,
which will result in arcing during electroporation. The efficiency of transformation increases a great deal if the DNA is ethanol precipitated, washed with 70% ethanol, and
resuspended in sterile water before use for transformation. This procedure decreases the
likelihood of arcing.
The first time a preparation of competent bacteria is used, positive and negative controls
should be included to determine the transformation competence and efficiency of the
bacteria, and to ensure there is no background contamination of drug-resistant bacteria.
These controls can usually be omitted during subsequent use of the preparation of bacteria
but should be repeated if no transformants result or if the transformants contain plasmids
of incorrect structure.
15. Transfer the cells and DNA mixture to the chilled electroporation cuvette, and tap
the cuvette to ensure the cell suspension covers the cuvette bottom.
16. Incubate the cuvette containing DNA and bacteria 10 min on ice (optional).
This optional 10-min incubation will usually increase the transformation efficiency twofold.
17. If using XL1 blue bacteria: Add 20 µl of 100 mg/ml IPTG and 40 µl of 25 mg/ml
Xgal to each plate and spread evenly.
The plates can be prepared during the 10-min incubation in step 16 or during the 30- to
60-min incubation of step 20. Keep the plates at room temperature. If the plates are wet,
incubate at room temperature, with the lids askew, until the plates are dry.
18. Dry the outside of the electroporation cuvette thoroughly with a Kimwipe and place
in the electroporator cuvette holder. Initiate an electrical pulse of 25 µF, 1.7 or 2.5
kV, 200 W with a time constant >4.1 (close to 5.0 is ideal).
19. Immediately add 1 ml SOC medium and use a sterile Pasteur pipet to transfer mixture
to a 15-ml polystyrene tube.
Transformation
of E. coli by
Electroporation
Using SOC medium instead of LB medium during this expression step increases the
transformation efficiency two-fold.
A.3N.2
Supplement 21
Current Protocols in Immunology
20. Incubate bacteria 30 to 60 min at 37°C with gentle rotation or shaking.
21. Plate bacteria on LB plates containing the appropriate antibiotic. Incubate plates
overnight at 37°C.
Usually it is best to plate at several concentrations because the number of colonies that
will result is unknown. Unplated bacteria can be stored at 4°C and plated at a later time
(≤1 week).
Colonies become visible in ∼14 hr. Colonies appearing after 24 hr represent contaminants.
XL1 blue colonies with inserts should be white if plated in the presence of IPTG and Xgal;
colonies without inserts will be blue.
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2; for suppliers, see APPENDIX 5.
LB plates
Per liter:
10 g tryptone
5 g yeast extract
5 g NaCl
1 ml 1N NaOH
15 g agar or agarose
Add water to 1 liter and autoclave. Pour plates with 32 to 40 ml medium (25 plates
per liter). Store at 4°C.
Plates without antibiotic can be stored indefinitely as long as they do not dry out.
SOC medium
2% (w/v) tryptone
0.5% (w/v) yeast extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
20 mM glucose
Store at 4°C.
Sterile filtered water
Rinse an 0.45-µm filter with 50 to 100 ml distilled water and discard the water. Filter
required volume of water using the rinsed filter. Store at 4°C.
Prerinsing the filter is required to remove the detergent from the filter; residual detergent
will interfere with bacterial viability.
The quality of water is critical for the success of the electroporation; the water must have
very low conductivity so it cannot be prepared by autoclaving.
Common
Immunologic
Techniques
A.3N.3
Current Protocols in Immunology
Supplement 21
COMMENTARY
Background Information
See Commentary in CPMB UNIT 1.8 for a discussion of transformation by electroporation.
Critical Parameters
The most frequently encountered problem
with electroporation is arcing in the cuvette,
usually caused by a medium that is too conductive. With the conditions described above, a
conductivity ≥10 meq will arc. Excessive conductivity can result from several sources. Excess salt may be present in the preparation of
bacteria. This can be caused by insufficient
washing of cells, by resuspending cells in a
buffer with too high an ionic strength, or by
lysis of cells in the preparation. A second source
of excess salt is the DNA preparation or ligation
mixture used for transformation. Ethanol precipitating the DNA, followed by washing in
ethanol and resuspending in distilled water, will
usually eliminate this problem. Arcing can also
be caused by a failure to adequately cool the
electroporation cuvette.
If transformants are not obtained using this
method several factors should be considered.
As noted above, arcing caused by excessive
conductivity is a frequent problem. When arcing occurs, transformation frequency falls precipitously. A second issue is the quality of the
recipient cells. Care must be taken to harvest
cells in early- to mid-log growth during preparation; rapidly growing cells seem to be best for
transformation by electroporation. Each new
batch of cells should be tested with a plasmid
of known concentration and quality to be certain that the cells are transformation competent.
Improper storage of the cells will also reduce
transformation efficiency; storage at −70°C is
preferred. Because electroporation is so effi-
cient, it is also easy to have extraneous DNA
contamination that interferes with the ability to
obtain the desired transformants. Therefore,
appropriate negative controls should always be
done.
The quality of the DNA used for transformation is also an important consideration. Contamination of DNA with substances such as
SDS, phenol, or cesium chloride (CsCl) can
severely inhibit transformation. Agarose gel
analysis can be used to confirm that the DNA
is intact and of the expected concentration. The
recipient strain must be selected to ensure that
there are no restriction barriers to the expression of the transforming DNA.
Manufacturers of apparatuses used for electroporation furnish literature which is very
helpful in troubleshooting.
Anticipated Results
Electroporation of E. coli should result in
transformation efficiencies of 5 × 108 transformants per milligram DNA.
Time Considerations
Washing and concentrating bacterial cells
takes ∼1 hr. Electroporation takes a few minutes; growth and plating of transformed cells
takes ≤90 min.
Literature Cited
Dower, W.J., Miller, J.F., and Ragsdale, C.W. 1988.
High efficiency transformation of E. coli by high
voltage electroporation. Nucl. Acids Res.
16:6127-6145.
Contributed by Sherie L. Morrison
University of California Los Angeles
Los Angeles, California
Transformation
of E. coli by
Electroporation
A.3N.4
Supplement 21
Current Protocols in Immunology