G418 selection and isolation of BT20 transfectant clones
1. Prepare G418 at a concentration of 100 ug/ml in MEM with 10% fbs.
Change the G418 media every 3 to 4 days (every Friday and Monday/Tuesday works
well).
2. Culture the cells in selection medium until colonies are visible.
3. Mark colonies on bottom of each plate.
Hold each plate up to the light at an angle and circle visible colonies on the bottom of
the plate with marker. Check under microscope to see that whole colony is within
circled area.
4. Grow up enough colonies on each plate for selection (5 to 40 colonies per plate?).
5. Prepare and label 24-well cell culture dishes to receive the new clones.
Minimize the amount of media added to each well. It may be useful to use 50%
conditioned medium that is removed from a more densely populated culture and sterile
filtered. Also, try supplementing media with 20% fbs rather than 10% to promote
growth.
6. Select individual colonies and transfer to separate wells.
a. Pour cloning disks into sterile 6cm2 dish. Add approx 1 ml trypsin to soak disks.
b. Remove media from 10cm2 plate (plate with transfectant clones). Wash with 10
ml PBS. Add approx 5 mls PBS to plate and tilt plate so that half is covered with
PBS and half is not (place edge on another 6cm2 plate to tilt). It may be useful to
draw a line through bottom of the plate separating top half from bottom half.
c. Place about 5 cloning disks soaked with trypsin onto separate colonies.
d. Incubate to complete trypsinization. Cloning disk package recommends 3 to 10
minutes whereas I worked as quickly as possible and did not wait between setting
the last cloning cylinder on a colony and picking the first colony.
e. Dip tip of forceps in ethanol and flame sterilize. Select colony by picking up edge
of cloning disk and gently scraping up colony. Transfer to new well and gently
shake the cloning disk in the media to loosen the cells from the disk. Leave
cloning disk in well with cells and media.
f. Repeat step “e” until all colonies are selected. Work quickly with top half of plate
so that colonies do not dry out. Once top half of plate is finished with selection,
rotate the plate so that the bottom half that was covered in PBS is now at the top.
Select colonies from this half.
g. Grow cells in 24-well plates. When ready to be transferred to 12-well plates,
trypsinize as usual and leave cloning disk behind in 24-well plate. After growing
up in 12-well plates, transfer to 6-well, then 6cm2, then 10cm2, and so on. Freeze
stock of cells and characterize.