1 |
Remove medium from 96-well plate and wash each well with 100ul of PBS, using the 8 channel pipette. |
2 |
Add 40 ul trypsin-EDTA to each well and leave the plate at 37C for 5 to 10 min. |
3 |
While trypsinizing cells, prepare 2X freezing medium in a round-bottom 96-well plate. Add 60ul 2X freezing medium per well and mark this plate the same way as the original plate. Keep the new plate at 4C. |
4 |
Add 60 ul Es+Lif medium to each well of the original plate and disperse cells by pipetting up and down aprox. 7 times, using the multichannel pipette. |
5 |
Wrap the new plate with parafilm and place it on dry ice for 30 min. Transfer the replica plate to -80C freezer. |
6 |
Add 150ul Es+Lif per well to the original plate. You can use Es without Lif at this point. |
7 |
After 2-3 days you have to expand these clones. |
|
|
| |
|
