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Transformación de E Coli Por Electroporación Morrison2001
Transformación de E Coli Por Electroporación Morrison2001
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
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 cient, it is also easy to have extraneous DNA
See Commentary in CPMB UNIT 1.8 for a dis- contamination that interferes with the ability to
cussion of transformation by electroporation. obtain the desired transformants. Therefore,
appropriate negative controls should always be
Critical Parameters done.
The most frequently encountered problem The quality of the DNA used for transfor-
with electroporation is arcing in the cuvette, mation is also an important consideration. Con-
usually caused by a medium that is too conduc- tamination of DNA with substances such as
tive. With the conditions described above, a SDS, phenol, or cesium chloride (CsCl) can
conductivity ≥10 meq will arc. Excessive con- severely inhibit transformation. Agarose gel
ductivity can result from several sources. Ex- analysis can be used to confirm that the DNA
cess salt may be present in the preparation of is intact and of the expected concentration. The
bacteria. This can be caused by insufficient recipient strain must be selected to ensure that
washing of cells, by resuspending cells in a there are no restriction barriers to the expres-
buffer with too high an ionic strength, or by sion of the transforming DNA.
lysis of cells in the preparation. A second source Manufacturers of apparatuses used for elec-
of excess salt is the DNA preparation or ligation troporation furnish literature which is very
mixture used for transformation. Ethanol pre- helpful in troubleshooting.
cipitating the DNA, followed by washing in
ethanol and resuspending in distilled water, will Anticipated Results
usually eliminate this problem. Arcing can also Electroporation of E. coli should result in
be caused by a failure to adequately cool the transformation efficiencies of 5 × 108 transfor-
electroporation cuvette. mants per milligram DNA.
If transformants are not obtained using this
method several factors should be considered. Time Considerations
As noted above, arcing caused by excessive Washing and concentrating bacterial cells
conductivity is a frequent problem. When arc- takes ∼1 hr. Electroporation takes a few min-
ing occurs, transformation frequency falls pre- utes; growth and plating of transformed cells
cipitously. A second issue is the quality of the takes ≤90 min.
recipient cells. Care must be taken to harvest
cells in early- to mid-log growth during prepa- Literature Cited
ration; rapidly growing cells seem to be best for Dower, W.J., Miller, J.F., and Ragsdale, C.W. 1988.
transformation by electroporation. Each new High efficiency transformation of E. coli by high
voltage electroporation. Nucl. Acids Res.
batch of cells should be tested with a plasmid
16:6127-6145.
of known concentration and quality to be cer-
tain that the cells are transformation competent.
Improper storage of the cells will also reduce
Contributed by Sherie L. Morrison
transformation efficiency; storage at −70°C is
University of California Los Angeles
preferred. Because electroporation is so effi- Los Angeles, California
Transformation
of E. coli by
Electroporation
A.3N.4
Supplement 21 Current Protocols in Immunology