University of Texas at San Antonio
SOP No.: 1. ver 1.0
Effective Date: 5/9/08
Page 1 of 4
Title: Generation of Electrocompetent E.coli and transformation procedure
Approved:
Author: Jeff Barker
Karl Klose
UTSA PI
Date: 6/17/2008
1. INTRODUCTION
The generation of electrocompetant E.coli is an essential component of all cloning
procedures done in the laboratory. Strains of E.coli are used to propagate and store
plasmids containing any gene of interest.
2. PURPOSE
This protocol describes the process of generating competant E.coli bacteria and the
electroportion of DNA into the competent cells. Plasmid DNA can be successfully
transformed into competent bacteria and is required for propagating and storing
plasmid DNA. Ligation reactions can be transformed into competent E.coli for the
use of screening potential clones as well. The proper storage and freezing of plasmid
DNA in competent E.coli allows for an infinite source of all cloned DNA.
3. RESPONSIBILITY
Research Technologist: performs the SOP
Technical Specialist: reviews the data, calculations and interpretations
Principal Investigator: reviews the data and interpretations
4. SCOPE
This procedure is used by the UTSA team under the direction of Dr. Karl Klose for
general cloning purposes.
5. PRECAUTIONS
Sterility and temperature maintenance are critical steps in successfully generating
competent bacteria for transformation. Be sure to use sterile media and tubes during
the entire procedure. To ensure that bacterial cell lysis is avoided bacteria must be
kept on ice or at 4C at all times.
6 MATERIALS AND EQUIPMENT
6.1 Sorval RC 5C Plus Centrifuge
6.2 Eppendorf table top centrifuge (Model 5415 D)
6.3 250 ml Centrifuge Bottles (sterile) (VWR, 21020-050)
6.4 50 ml Centrifuge Bottle (sterile) (VWR, 21009-320)
6.5 250 ml Erhlenmeyer Flasks (VWR, 89000-362)
1
University of Texas at San Antonio
SOP No.: 1. ver 1.0
Effective Date: 5/9/08
Page 2 of 4
Title: Generation of Electrocompetent E.coli and transformation procedure
Approved:
Author: Jeff Barker
Karl Klose
UTSA PI
Date: 6/17/2008
6.6 Sterile 1.5 ml centrifuge tubes (Biolink, BL3150LR)
6.7 Luria Broth (LB)
6.8 Nichipet EX Plus pepettes, calibrated annually (p10, p20, p200, p1000)
6.9 Drummond pipet-aid (Drummond, Portable Pipet Aid XP)
6.10 Sterile Serilogical pipettes (5 ml, 10ml, 25 ml)
6.11 37C shaking incubator (VWR, model 1545)
6.12 Sterile 5 ml glass tubes (VWR, 89000-496)
6.13 10% Glycerol (sterile)
6.14 Quick Freeze Apparatus
6.15 Dry ice
6.16 100% Ethanol
6.17 Bio-Rad 0.1 cm electrode gap cuvette
6.18 Bio-Rad Gene Pulsar II
6.19 Sterile 1 ml glass tubes (VWR, 89000-494)
7. PROCEDURE FOR GENERATION OF ELECTROCOMPETANT E.coli
The following procedure is designed to generate ~30 samples of electrocompetant
E.coli samples.
7.1 Luria Broth (LB) Media Preparation
7.1.1 Dissolve 5 gms NaCl, 10g Tryptone, 5 g Yeast extract in 1 L double
deionized water.
7.1.2 Autoclave media to sterilize.
7.2 Generate overnight liquid culture of E.coli
7.2.1 Inoculate 5 ml LB with E.coli frozen stock.
7.2.2 Incubate in 37C incubator overnight
7.3 Preparation of Electrocompetant E.coli
7.3.1 Inoculate two 250 ml LB cultures (500 ml flask) with your overnight
culture of bacteria (2.5 mls of overnight into each 250 ml of LB media in
each 500 ml Flask).
7.3.2 Grow Shaking (160 RPM) for 2-4 hours until turbid (OD600 of ~0.4).
7.3.3 Place three 100 ml bottles of sterile ddH2O and 12 ml 10% sterile glycerol
in an ice bucket.
7.3.4 Harvest the two 250 ml culture at 5000 rpm for 10 minutes at 4C in two
balanced sterile, 250 ml centrifuge bottles.
2
University of Texas at San Antonio
SOP No.: 1. ver 1.0
Effective Date: 5/9/08
Page 3 of 4
Title: Generation of Electrocompetent E.coli and transformation procedure
Approved:
Author: Jeff Barker
Karl Klose
UTSA PI
7.3.5
7.3.6
7.3.7
7.3.8
7.3.9
7.3.10
7.3.11
7.3.12
7.3.13
7.3.14
7.3.15
7.3.16
Date: 6/17/2008
Discard the supernatant, retain the pelleted cells, and keep cells on ice at all
times between centrifugations.
Thoroughly resuspend each pellet in 100 ml ice-cold, sterile H2O and pool
the two suspensions into one 250 ml centrifuge bottle.
Centrifuge at 5000 rpm for 10 minutes, 4C.
Discard the supernatant.
Resuspend the pellet in 100 ml sterile, ice-cold H2O.
Centrifuge at 5000 rpm for 10 minutes, 4C.
Discard the supernatant.
Resuspend in 10 ml 10% glycerol and transfer to a sterile 50 ml centrifuge
bottle.
Centrifuge at 5000 rpm for 10 minutes, 4C.
Discard all the supernatant.
Thoroughly resuspend the cell pellet into 1 ml sterile 10% glycerol.
Aliquot the suspension into the 40 ul volumes into labeled sterile 1.5 ml
centrifuge tubes and immediately add tube to Quick Freeze Apparatus.
7.4 Freezing of Electrocompetant E.coli
7.4.1 Fill the Quick Freeze Apparatus with dry ice and 100% Ethanol.
7.4.2 Place aliquoted tube into Quick Freeze Apparatus dry ice/ethanol bath.
Samples should freeze instantly.
7.4.3 Store Frozen tubes at -80C.
7.
PROCEDURE FOR ELECTROPORATING COMPETANT E. coli
7.4.4 Transforming Electrocompetant E.coli
7.4.4.1 Place 0.1 cm electrocuvette on ice
7.4.4.2 Thaw electrocompetant E.coli on ice
7.4.4.3 Add DNA of interest to 40 ul electrocompetant bacteria. (Commonly use
1-2ug of DNA)
7.4.4.4 Transfer sample to electrocuvette on ice.
7.4.4.5 Electroporate using Bio-Rad Gene Pulsar with the settings: 2.5 kV, 25
uF Capacitance, 200  (ohms) resistance.
7.4.4.6 Immediately add 1 ml of LB and transfer to a sterile 1 ml glass tube.
7.4.4.7 Incubate shaking at 150 rpm at 37C for 1 hour.
7.4.4.8 Spin down sample at 10,000 RPM in table top centrifuge for 1 min.
7.4.4.9 Resuspend pellet in 100 ul of LB and spread onto a LB agar plate
containing the appropriate antibiotic.
3
University of Texas at San Antonio
SOP No.: 1. ver 1.0
Effective Date: 5/9/08
Page 4 of 4
Title: Generation of Electrocompetent E.coli and transformation procedure
Approved:
Author: Jeff Barker
Karl Klose
UTSA PI
Date: 6/17/2008
7.4.4.10
In general terms, the success of the transformation can be
confirmed in multiple ways, including colony selection on plates
containing the appropriate selective antibiotic, pcr on plasmid minipreps, DNA sequencing etc.
8. QUALITY CONTROL
Restriction digests on mini plasmid preps to will verify the correct plasmid has been
transformed into the Ecoli .
9. REFERENCES
None
10. CALCULATIONS AND FORMULAS
None
11. GLOSSARY
 ml- milliliter
 cm- centimeter
 LB- Luria Broth
 Kv- kilovolts
 RPM- revolutions per minute
 C- centigrade
12. APPENDICES
None
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