If I have 2.8*10^6 cells (2,800,000)
and I set up serial dilution as follow
10ul from the above + 90ul medium (100ul = 280,000)
10ul from the above + 90ul medium (100ul = 28,000)
10ul from the above + 90ul medium (100ul = 2800)
Now I have 2800 cells in 100ul (1ul has 28 cells)
Can I add 2700ul medium to the above 100ul (2800 cells) to make 1cell/ul?
and add 200ul from the above 3000ul to 19,800ml medium now for every 200ul there is 1cell?
Will this make a successful single cell dilution?
I think you made a mistake in the last step.
200µl contains 200 cells. Your final volume is 20ml. So each 100µl contains 1 cell.
Hi Xin,
Basically, it will be worked. However, these are all theoretical value. If you recloned the cells into more wells of microplate, you may have more chance to get single cell in single well (e.g. 100 cells to 200 wells or more...). Of course, you may get some wells with multiple clones or none. It well depend on the level of limit dilution.
Best,
Hi!
another possibility is this protocol (see below). You will have to check in which wells only one cell was seeded. I checked after a couple of days in which wells one cluster of cells (originating from one cell by cell division) was visible.
• add 100 µl medium to all wells except well A1 of a 96 well plate
• in well A1: 200 µl with 1-2x 10E4 cells/ml
• 1/2 dilution series: resuspend 100 µl down the column (wells A1-H1)
• discard 100 µl from well H1 (final volume 100 µl)
• add 100 µl to all wells in column 1 (wells A1-H1)
• 1/2 dilution series: resuspend 100 µl down the rows (1-12)
• discard 100 µl from row 12 (wells A12-H12, final volume 100 µl)
• add 50 – 100 ml medium or conditioned medium
Should we perform centrifugation after trypnisization to replate cells after then in 96 wells plate!
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