OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
STGCL.SWP.71.1
School/ Divisional Unit
St George Clinical School
Initial Issue date
29/04/10
Current version
1.0
Current Version
Issue date
29/04/10
Next review date
29/04/2012
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this form.
Safe Work Procedure Title and basic description
Title: Plasmid DNA Transfection of mammalian cells in culture using Lipofectamine™ 2000, Lipfectamine Reagent
and Lipofectamine Plus Reagent
Description: To introduce nucleic acids into eukaryotic cells for expression of a particular mammalian gene.
Associated risk assessment title and location: RA Plasmid DNA Transfection of mammalian cells
Describe the activity or process
Transfection of cells in culture typically involves opening transient pores or "holes" in the cells, to allow the uptake
of material. Transfection can be carried out using calcium phosphate, by electroporation, or by mixing a cationic
lipid such as lipofectamine Plus Reagent or lipofectamine 2000. The material produces liposomes, which fuse with
the cell membrane and deposit their cargo inside.
Procedure:
1. Adherent cells: One day before transfection, plate 0.5-2 x 105 cells in 500 µl of growth medium without
antibiotics (in a 24 well plate) so that cells will be 90-95% confluent at the time of transfection.
Suspension cells: Just prior to preparing complexes, plate 4-8 x 105 cells in 500 µl of growth medium without
antibiotics (in a 24 well plate).
1.
24 hr after seeding, transfection is conducted with the following volumes:
Plate
Surface area per
well
24 well
2 cm2
Volume of
plating medium
500 µl
DNA Transfection
DNA
lipofectamine 2000
0.8 µg (in 50µl SFM)
2ul (in 50µl SFM)
2. For each transfection sample, prepare complexes as follows:
a. Dilute DNA in 50 µl of Opti-MEM® I Reduced Serum Medium without serum (or other medium without serum).
Mix gently.
b. Mix Lipofectamine™ 2000 gently before use, then dilute the appropriate amount in 50 µl of Opti-MEM® I
Medium. Incubate for 5 minutes at room temperature (RT). Note: Proceed to Step c within 25 minutes.
c. After the 5 minute incubation, combine the diluted DNA with diluted Lipofectamine™ 2000 (total volume = 100
µl). Mix gently and incubate for 20 minutes at RT (solution may appear cloudy). Note: Complexes are stable for 6
hours at RT.
3. Add the 100 µl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and
forth.
4. Incubate cells at 37°C in a CO2 incubator for 18-48 hours prior to testing for transgene expression. Medium may
be changed after 4-6 hours.
5. For stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after
transfection. Add selective medium (if desired) the following day.
Note: DNA and lipofectamine 2000 amounts may be varied to optimize results.
*SFM-serum free medium
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Page 1 of 4
Safe Work Procedure
Uncontrolled document when printed
Date Effective: 01/01/2007
Current Version: 1.2, 15/08/2007
Procedure: Transfections with Lipofectamine™ Reagent
1. One day before transfection, plate 2-6 x 104 cells in 500 µl of growth medium (with the usual amount of serum)
without antibiotics (in a 24 well plate) so that cells will be 50-80% confluent at the time of transfection.
2. For each transfection sample, prepare complexes as follows:
a. Dilute 0.2-0.4 µg DNA in 25 µl of Opti-MEM® I Reduced Serum Medium (or other medium) without serum. Mix
gently.
b. Mix Lipofectamine™ gently before use, then dilute 0.5-5 µl in 25 µl of Opti-MEM® I Medium (or other medium)
without serum. Mix gently.
c. Combine the diluted DNA with diluted Lipofectamine™ (total volume = 50 µl). Mix gently and incubate for 1545 minutes at RT (solution may appear cloudy). Note: Complexes are stable for 6 hours at RT.
d. For each transfection, add 0.15 ml of Opti-MEM® I Medium to the tube containing the complexes (total volume =
200 µl). Mix gently.
3. Remove the growth medium from cells and replace with 0.2 ml of growth medium without serum. Add the 0.2 ml
of diluted complexes (from Step 2d) to each well. Mix gently by rocking the plate.
4. Incubate cells at 37°C in a CO2 incubator for 2-24 hours. We recommend starting with 5 hours.
5. Add 0.4 ml of growth medium containing 2X the normal concentration of serum without removing the
transfection mixture. Note: If toxicity is observed after transfection, replace medium with fresh, complete medium
(with normal amount of serum).
6. For transient transfection: Test for transgene activity 24-72 hours post-transfection as appropriate for your cell
type and expression vector.
For stable transfection: Passage cells at a 1:10 dilution into selective medium 72 hours post-transfection.
Procedure: Enhancing Transfections with Lipofectamine™ Reagent
1. The day before transfection, plate cells in 24-well plates so that they are 50-80% confluent the day of transfection.
At the time of plating and during transfection, avoid antibiotics.
2. Pre-complex the DNA with the Plus™ Reagent: Dilute 0.4 µg DNA into 25 µl dilution medium without serum
(Dulbecco’s Modified Eagle Medium or similar medium). Mix Plus™ Reagent before use. Add 4 µl Plus™ Reagent
to diluted DNA, mix again, and incubate at RT for 15 minutes.
3. Dilute 1 µl Lipofectamine™ Reagent into 25 µl dilution medium without serum in a second tube; mix.
4. Combine pre-complexed DNA (from step 2) and diluted Lipofectamine™ Reagent (from step 3); mix and incubate
for 15 minutes at RT.
5. While complexes are forming, replace the medium on the cells with 0.2 ml of cell growth medium without serum.
Note: It is possible to include serum in the cell growth medium at this step.
6. Add the DNA-Plus™-Lipofectamine™ Reagent complexes (from step 4) to each well of cells containing fresh
medium. Mix complexes into the medium gently; incubate at 37°C at 5% CO2 for 3 hours.
7. After the 3 hours incubation, increase volume of medium to normal volume; add serum to bring the final
concentration to that of normal growth medium. If necessary to maximize cell growth, replace the medium
containing the complexes with fresh, complete medium after 3 h incubation or the day after transfection.
8. Assay cell extracts or stain cells in situ for reporter gene activity 24-48 hours after the start of transfection,
depending on cell type and promoter activity.
For stable transfection: Passage cells at a 1:10 dilution into selective medium 72 hours post-transfection.
List all resources required including plant, chemicals,
personal protective clothing and equipment, etc
Biological Safety Cabinet
Tissue culture lab coat
Latex Gloves
Tissue culture medium
Tissue culture equipment (plates, pipettes, pipette gun, 24 well plate)
70% ethanol
Lipofectamine 2000
Lipofectamine Reagent
Lipofectamine Plus Reagent
Selective antibiotics
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Page 2 of 4
Safe Work Procedure
Uncontrolled document when printed
Date Effective: 01/01/2007
Current Version: 1.2, 15/08/2007
List potential hazards and risk controls including
specific precautions required
1- Working with human cell lines carries a potential biohazard risk.
 Avoid exposure to cell cultures by wearing personal protective equipment (long sleeve gown which ties up
at the back and latex gloves).
 Clean the biological safety cabinet with 70% ethanol thoroughly after use to remove any potential biohazard
within this sterile environment (see Biological Safety Cabinet SWP: STGCL.SWP.31.1
 Turn on UV lamp for at least 20 minutes after use to kill any living organisms within the cabinet.
2- Selective antibiotic hazards
a-Hygromycin
Potential Health effects
Eyes Risk of serious damage to eyes.
Skin Toxic in contact with skin. Severe skin irritation.
Inhalation Toxic by inhalation.
Ingestion Toxic if swallowed.
 Correct PPE (Gloves, lab coat, face mask and glasses) and lab safety training.
 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice
 After contact with skin, wash immediately with plenty of water
 In case of accident or if you feel unwell seek medical advice immediately
First Aid: For advice, contact a Poison Information Centre on 13 11 26 (Australia Wide) or a doctor (at once).
b-Geneticin
Potential Health Effects:
Eye: Can cause moderate irritation, tearing and reddening, but not likely to permanently injure eye tissue.
Skin: Can cause moderate skin irritation, defatting, and dermatitis. Not likely to cause permanent damage.
May cause sensitization.
Inhalation: Can cause moderate respiratory irritation, dizziness, weakness, fatigue, nausea and headache.
May cause allergic respiratory reaction.


Correct PPE (Gloves, lab coat, face mask and glasses) and lab safety training.
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice
First Aid: For advice, contact a Poison Information Centre on 13 11 26 (Australia Wide) or a doctor (at once). If
swallowed, do not induce vomiting.
List emergency shutdown instructions
First Aid: For advice, contact a Poison Information Centre on 13 11 26 (Australia Wide) or a doctor (at once).
List clean up and waste disposal requirements
Refer to SWP for Safe Work Procedure for Waste Disposal at St George Clinical School (STGCL.SWP.2.1)
List legislation, standards and codes of practice used
in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3-2002. Safety in laboratories. Part 3: Microbiological aspects and containment facilities.
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Page 3 of 4
Safe Work Procedure
Uncontrolled document when printed
Date Effective: 01/01/2007
Current Version: 1.2, 15/08/2007
Supervisory approval, training, and review
Supervisor: Prof Beng Chong (Medicine)
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Plant custodian: Prof Beng Chong
Signature
List competency required – qualifications, certificates, licensing, training - eg course or instruction:
Induction into the lab,
UNSW PC2 training
Training in this SWP.
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Page 4 of 4
Safe Work Procedure
Uncontrolled document when printed
Date Effective: 01/01/2007
Current Version: 1.2, 15/08/2007