382
Related Techniques
DNA - Plasmid DNA-containing genes to be inserted into host genome DNA
Procedure
Pretreatment 1. One day before lipofection, harvest cells from a culture in which the
cells are growing in a semiconfluent state, and seed the cells into a new
flask with 3.0 ml culture medium at a density of 10 6 cells/ml.
2. Culture for 1 or 2 days.
3. Remove the medium without disturbing the cells, leaving 0.5 ml culture medium over the cells.
4. Add 3.0 ml serum-free culture medium.
5. Gently swirl the flask, let it sit for 10 min, and then swirl the flask
again.
Lipofection 1. Add 50 fll DNA-Lipofectin mixture to the flask.
2. Add sufficient FBS after 24 h in order to obtain a final concentration
(usually 10 %).
3. Allow the cells to remain in culture for another day.
4. The cells are then ready for a transient assay.
Results
Using this method, Malone et al. (1989) were able to transfect cultured
Drosophila cells with Photinus pyralis luciferase mRNA.
Subprotocol 4
Electroporation Method
Materials
Equipment - Plastic culture flasks
- Pasteur pipettes
- Electroporation apparatus (e.g., Gene Pulser II; Bio-Rad)
- Cuvette electrode
Solutions - Culture medium
DNA - Plasmid DNA-containing genes to be inserted into host genome DNA
Procedure
1. Seed the cells at a density of approximately 10 6 cells/mIl or 2 days
before electroporation. The interval between cell seeding and
electroporation is sometimes critical. This interval should be predetermined.
2. Harvest the cells, and wash twice with serum-free culture medium.
Related Techniques
DNA - Plasmid DNA-containing genes to be inserted into host genome DNA
Procedure
Pretreatment 1. One day before lipofection, harvest cells from a culture in which the
cells are growing in a semiconfluent state, and seed the cells into a new
flask with 3.0 ml culture medium at a density of 10 6 cells/ml.
2. Culture for 1 or 2 days.
3. Remove the medium without disturbing the cells, leaving 0.5 ml culture medium over the cells.
4. Add 3.0 ml serum-free culture medium.
5. Gently swirl the flask, let it sit for 10 min, and then swirl the flask
again.
Lipofection 1. Add 50 fll DNA-Lipofectin mixture to the flask.
2. Add sufficient FBS after 24 h in order to obtain a final concentration
(usually 10 %).
3. Allow the cells to remain in culture for another day.
4. The cells are then ready for a transient assay.
Results
Using this method, Malone et al. (1989) were able to transfect cultured
Drosophila cells with Photinus pyralis luciferase mRNA.
Subprotocol 4
Electroporation Method
Materials
Equipment - Plastic culture flasks
- Pasteur pipettes
- Electroporation apparatus (e.g., Gene Pulser II; Bio-Rad)
- Cuvette electrode
Solutions - Culture medium
DNA - Plasmid DNA-containing genes to be inserted into host genome DNA
Procedure
1. Seed the cells at a density of approximately 10 6 cells/mIl or 2 days
before electroporation. The interval between cell seeding and
electroporation is sometimes critical. This interval should be predetermined.
2. Harvest the cells, and wash twice with serum-free culture medium.