Sack: Plating Subclones for Flourescent Screening

Plating subclones for fluorescent screening For every 12 colonies picked, you will need: 21ml appropriate maintenance media
1 TC-treated 12-well plate
1.5 8 well chambered coverglass slides
1 10ml serological pipet
serological pipetor
15 200 ul pipet tips (3 rows of 12)
200 ul pipetor
waste box for pipet tips
pen

1. Choose 12 wells on the 96-well plate that contain one fluorescent colony. Choose from the lowest density plate first.

2. Remove media from selected wells, rinse once with 200ul PBS, then add 50 ul trypsin. Watch to see when cells detach.

3. While cells are trypsinizing: put 1 ml media in each well of 12-well plate put 0.5ml media into each of the 12 chambers mark each well and chamber with name of a chosen well from the 96 well plate

4. Once cells detach, to each well add 200ul of media triturate 5x to suspend cells and remove 200ul suspension plate one drop of suspension in appropriately marked chamber place the rest of the cells in appropriate well of 12 well plate

5. Add 200 ul media to picked wells of 96-well plate, this makes a backup from straggling cells.

6. Place cells in incubator.

7. Check chambered slides for fluorescence 1-2 days later.

8. Plate upto 6 good colonies in T-75, using all cells in the 12 well plate when confluency is 50-90%.

9. Freeze when T-75 is 50-90% confluent.

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