When cells are medium-light, replace medium with 0.5 g/L G418 (= 0.5 mg/mL)

Next day, replace with medium with 1 g/L (=1 mg/mL)

Next day, 2 g/L (=2 mg/mL)

Solution is 50 mg/mL

Dilute 10 uL per mL to get 0.5 mg/mL

Dilute 20 uL per mL to get 1 mg/mL

Dilute 40 uL/mL to get 2 mg/mL

1. Make a death curve. This is to determine the minimal concentration of antibiotic that kills cells that do not have the resistance gene.
2. transfect the cells with the plasmid containing the gene that you want to express in the cells (see Amaxa protocol).
3. incubate the cells for about 48 hours after transfection so that the gene is expressed, and the resistence gene is expressed.
4. add the appropriate dose of antibiotic (this is the selection step). The cells that do not contain the resistance gene (i.e. that were not successfully transfected) will die. Change the medium every 2 or 3 days to remove dead cells. Replace medium with fresh medium containing the antibiotic.
5. split the cells as needed. Use medium with antibiotic. Make some coverslips to see if the cells are expressing the gene of interest.

After ~2 weeks any cell without the transgene and selectable marker should have died. The remaining cells are a mixed population – different cells might express different levels of the transgene. These can be used for preliminary experiments.

Making a clonal cell line

A Clonal cell line contains cells that are derived from a single cell, and thus are a clone. They will all be genetically identical; this can be very helpful for experiments.

a. Trypsinize your 'mixed population' of cells; keep some growing in a flask and freeze some in case your cloning does not work the first time and you need to repeat the procedure.

b. take 50ul of the trypsinized cells, dilute again into 1 ml medium. Add 10 -150 ul of this dilute solution of cells to 100mm dishes containing growth medium. Look under the tissue culture scope. The cells should be very dilute – one or just a few cells per field of view. If they are too dense or dilute, adjust accordingly. You want each individual cell to land on the plastic dish and grow into a colony of cells that is separate from other colonies on the dish.

c. wait about 1 week for the colonies to grow.

d.isolate individual colonies. These are potential clonal cell lines.

• 1. prepare cloning rings by smearing a small amount of grease on one end; make about 10. Have sterile forceps ready; have trypsin ready. Have a 24 well plate ready with some medium in the number of wells that you are planning to use.
• 2. remove medium from dish.
• 3. rinse with PBS--; leave a little bit so the cells don't die.
• 4. tilt the dish to see the colonies; place a cloning ring over nice large and well separated colonies. Press down firmly. Don't let the ring slide around. Add ~100ul of trypsin to each ring; wait a few mins. Pipette cells up and down (in the ring) add the released cells to one well of a 24 well plate. Each well now has a colony of cells – a potential cell line. When they grow, split each well – place some cells in a well of a six well plate to grow some more and put some in a Matek dish – be sure to label carefully so you know which is which. When the cells in the Matek dish are grown – check them in the fluorescence microscope. If they all have the same level of fluorescence and are healthy looking, they are a potential line (which you can further characterize with a Western Blot to quantify the level of expression).
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Toss any cell lines that are not healthy or have too much or too little fluorescence.