Biology Reference
In-Depth Information
1.
Thaw one cryovial of feeder cells at 37
C. Dilute cells in 10 mL of feeder
medium. Spin 5 min at 120
g
. Decant supernatant and resuspend cells in
25 mL of feeder medium. Aliquot 1 mL of cell suspension into each well of a
24-well dish. Repeat this step for 10 or more (depending on the number of colonies
that are to be picked) 24-well dishes. Let fibroblasts adhere and spread overnight.
°
2.
Just before starting to pick the colonies, change medium of 24-well dishes to
ES Medium.
3.
With a 5-mL pipet, transfer 2 drops of trypsin into each well of a 96-well
microtiter dish. Warm at 37
°
C.
4.
Using a stereomicroscope under a laminar flow hood, gently scrape a colony
with a P200 pipet equipped with a sterile yellow tip. Suck the cell aggregate in a
maximum volume of 10
L and transfer it in a 96-well filled with trypsin. Repeat
this step for 12 colonies (
see
Note 8
).
µ
5.
Incubate the 96-well dish at 37
C for 5 min. Open one 24-well dish with feeder
and the 96-well dish under the hood.
°
6.
With a fresh, sterile yellow tip collect about 100
L of ES Medium from a feeder
cells-containing well. Add it to a trypsin well. Dissociate the trypsinized colony
by gentle pipeting. Carefully transfer the cell suspension to the same well from
which ES Medium was taken. Make a few air bubbles to mark the well. Repeat
this step for all trypsinized colonies (
see
Note 8
).
µ
7.
Repeat
steps 4-6
until enough colonies have been picked.
8.
The day after, change to fresh ES Medium all wells that received a colony.
9.
Wait 3-4 d for ES cells to expand. As soon as the number and size of colonies are
suitable for sibling, cells from each well can be frozen.
10.
Mark cryovials with progressive numbers. Mark the same numbers on the bottom
of each well to be passaged.
11.
Set an empty cryobox into dry ice. Put labeled cryovials into the holding device.
12.
Wash all marked wells of a 24-well dish with 1 mL PBS. Add 2 drops of trypsin
into each well and incubate 5 min at 37
°
C.
13.
Collect 900
L of freezing medium with a P1000 equipped with a fresh, sterile
blue tip. Thoroughly resuspend trypsinized cells of one well by gentle pipeting.
Retrieve only about 600 mL of cell suspension and transfer it into the cryovial
with the corresponding number. Place the cryovial into the box in dry ice. Repeat
this step for all trypsinized wells.
µ
14.
Fill each well to maximum with feeder medium to dilute DMSO, which can even-
tually be toxic for cells.
15.
Repeat steps 12-14 until all clones have been frozen.
16.
The day after freezing, it is extremely important to change medium to feeder medium.
3.3.4. Identification of Homologous Recombinants
1.
When medium inside a well turns to yellow, wait an additional day. Then discard
the medium and add 500
µ
L of Lysis buffer. Keep at 37
°
C until all wells have
been kept with Lysis buffer for at least one night.
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