Get 1. Cell Cloning By Serial Dilution In 96 Well Plates. Protocol ...

To perform serial dilution, for example, 1 ml of the starting sample is added to 9 ml of Dilution Blank Tube 1. This is then followed by the same procedure, where 1 ml from Tube 1 is added to 9 ml of Tube 2, 1 ml from Tube 2 is added to 9 ml from Tube 3, and so on until the desired concentration is reached.

Typically, 20,000 – 75,000 cells are seeded per well in Oris™ and Oris™ Pro 96-well plates, and 2,500 – 10,000 per well in Oris™ Pro 384-well plates.

For any cell type you can use a minimum of 100ul to a maximum of 200ul culture medium per well in a 96-well plate.

You have 3.348 x 10^6 cells. So if you need to seed 10000 cells per well, then for 100 wells you will need 10000 x 100 = 1 x 10^6 cells for a 96-well plate. If you seed cells at 100 ul per well, then you will need a total volume of 100 x 100 = 10000ul = 10 ml for 96-well plate.

You have 3.348 x 10^6 cells. So if you need to seed 10000 cells per well, then for 100 wells you will need 10000 x 100 = 1 x 10^6 cells for a 96-well plate. If you seed cells at 100 ul per well, then you will need a total volume of 100 x 100 = 10000ul = 10 ml for 96-well plate.

Plate 200 µL of cell culture (i.e., 10,000–50,000 cells) into the wells of the sterile 96-well cell culture plate.

Limiting dilution cloning (LDC) We recommend performing limiting dilution by targeting 0.8 cells/well, which requires you to resuspend the transfected cells (post-counting) at a density of 8 cells/mL in complete growth medium, then transferring 100 μL of this to each well of a 96‑well plate.

You can calculate your cell concentration using the following formula: Total cells/ml = (Total cells counted x Dilution factor x 10,000 cells/ml)/ Number of squares counted. Total cells/ml = (325 cells x 2 x 10,000 cells/ml)/ 5 = 130 x 104 cells/ml. Total cells in sample = 130 x 104 cells/ml x 5 ml = 650 x 104 cells.