Kevin Moses

G418 SELECTION

G418 selection;
I have found that a concentration of 1 mg/ml G418 cleanly selects between one dose of an insertion of pHSNEO; and wild-type. However there is some batch variation in the effective percentage of the drug. Therefore I recommend a pilot run with a known transformed stock and wild-type, at perhaps 0, 0.5, 1.0, 1.5 and 2.0 mg/ml G418. I also recommend running such controls in each experimental run.

1) G418 is made up as 100 mg/ml stock solution and stored at -20°C in 5 ml aliquots.

2) Yeast-glucose Drosophila medium is made by boiling the following for 5 minutes:

10% weight/volume dry yeast
10% weight/volume glucose (sucrose works also)
1.6% weight/volume agar
3 % volume/volume nipagen (‰tegosept, 10% w/v in ethanol)

3) Bring the food to ~ 50°C in a water bath.

4) Add the G418 with a dash of bromophenol blue (in water) and mix with a flea (the blue dye both demonstrates that the mixing is complete and marks the food as it will be poisonous to most flies).

5) Pour tubes or bottles in the normal way and store the food at 4°C.

6) Before use bring to room temperature and add a minimum quantity of wet yeast (so that the adults will completely consume it in two days.

7) Allow the adults two days on the food and then sub on.

8) If using heat-shock inducible vectors, raise the cultures once to 37°C for 30 minutes on the next day. This should suffice.