Picking Colonies After Selection

This can either be done with the naked eye or by placing a dissecting microscope (such as a Lietz M3B) into the laminar flow hood.

1. Colonies are ready for picking if they are large and well separated (usually 7-9 d after electroporation). For picking start by gently washing the plate twice with PBS.

2. After the second rinsing, leave a little of the PBS behind in the dish (about 1 mL) in order to keep the surface of the plate wet, therefore preventing the colonies from drying out.

3. Choose a colony to pick. The colony is optimal if it is neither too small nor too big, and contains nondifferentiated ES cells with characteristic morphology. Fill a P200 pipeter with approx 20 ||L PBS, and gently pour this over the colony thereby rinsing it. Retain about 5-10 |imL of the PBS in the pipet, and with this, try to "suck up" the colony from the bottom of the plate. Hold the pipet perpendicular (if picking with the naked eye) or at 45° (if picking under the dissecting microscope) to the surface of the plate, since this facilitates the lifting of the colony from the plastic.

4. Using the P200, transfer each individual colony into separate wells of a 96-well plate containing 50 | L trypsin in each well. In doing so, pipet the colony up and down several times in order to dissociate the cells. Be careful to avoid creating too many air bubbles.

5. When all the colonies from a plate have been picked and transferred to the trypsin, the 96-well plate is placed in a 37°C incubator for 5-10 min.

6. During this time a new gelatinized 96-well flat-bottom plate with medium (200 |L/well, containing the selection agent) is prepared.

7. Working row by row with a multichannel pipeter the cell-trypsin solution is transferred to the gelatinized plate.

8. Pipet thoroughly, without creating too many air bubbles, so as to promote the formation of a single cell suspension.

9. Return the plate to the 37°C incubator.

10. Change the media daily until the cells are ready to passage (80% confluency).

11. When passaging the cells, split them into two or three new plates. These can each be used for the preparation of DNA for genotype screening and for creating frozen stocks, which can then be used for thawing the required clones.

Was this article helpful?

0 0

Post a comment