Q:
A:
63
Don't use Anti-Clumping Agent in cloning medium (although it can be
present in the growth medium used for dilution prior to the addition of
cells to cloning medium, as it will be diluted).
How do I know I have cells in the final dilution for limited dilution cloning?
Assuming you are diluting to plate 1 cell/well in a 96-well plate, your
second-to-last dilution is designed to achieve 1,000 cells/mL, and
subsequently, the final dilution would be 200 μL of 1,000 cells/mL +
40 mL of cloning medium to give 5 cells/mL. Plating 200 μL per well
generates the desired 1 cell/well plating.
Procedure:
• Using the predicted 1,000 cells/mL dilution, seed 12 wells of a
flat-bottom 24-well plate with 8 μL of cells.
– If the initial cell count and subsequent dilution steps were
accurate, you should see ~8 cells/well on average.
• Count the total number of cells in the 12 wells and divide by 96
μL. Multiply by 1,000 to get the cell suspension concentration, and
use this corrected value to achieve your desired final dilution.
Example:
• If you count 65 cells in 96 μL, you have 65/96 x 1,000 = 677 cells/mL.
• Using the accurate cell count of 677 cells/mL, your final dilution
to achieve 5 cells/mL per 40 mL of cloning medium is:
5 cells/mL x 40 mL/677 cells/mL x 1,000 =
295 μL of 677 cells/mL + 39.7 mL of cloning medium.
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