Biology Reference
In-Depth Information
10 7 cells (the content of a
inserted. In an average transfection experiment, 2
×
full 10-cm Petri dish) are transfected with 30
µ
g of DNA.
1.
Using feeder medium, plate irradiated fibroblasts (about 9 cryovials) onto nine
10-cm tissue-culture dishes. Change to ES medium when cells are adherent
and spread.
2.
Linearize 30
g of the construct digesting with a single cutter enzyme (usually
Not I) that cleaves at the boundary between one homology arm and the vector.
Keep a final DNA concentration of at least 50 ng/
µ
µ
L. Incubate 1 h at the suitable
temperature, using 30 or more enzyme units.
3.
Extract the reaction mixture once with an equal volume of phenol/chloroform.
Spin at maximum speed for 5 min. Collect the supernatant and extract it once
with chloroform. Spin at maximum speed for 30 s. Save the supernatant.
4.
Add to the supernatant 1/10 of the volume of 3 M Na acetate and 2 volumes of
100% ethanol. Mix thoroughly. A white DNA precipitate particle must appear.
Using a yellow tip, transfer the precipitate in a sterile screw cap tube containing
1 mL of 70% ethanol/DDW. Keep in ice until needed.
5.
Wash ES cells 2 times with PBS. Add 1 mL of warm trypsin. Incubate 5 min at
37
C. After adding 2 mL of ES Medium, dissociate colonies by pipeting up and
down, avoiding the production of bubbles.
°
6.
Dilute cells to 10 mL and count them using a Burker's chamber. Cells with large
cytoplasm derive from the feeder layer and should thus be omitted from the count.
10 7 cells in a sterile 10-mL tube 5 min at 120 g . Resuspend 5 mL of PBS.
Repeat this step twice.
7.
Spin 2
×
8.
Spin again and resuspend in PBS to reach a final volume of 600
µ
L.
9.
Spin the DNA precipitate for 30 s. Discard the supernatant under sterile condi-
tions and let the pellet dry out for few min inside the hood. Resuspend DNA in
200
µ
L PBS.
10.
Mix the cell suspension with DNA. Using a Pasteur pipet, transfer the mixture
into a sterile cuvet for electroporation. Electroporate cells at 3
F and 0.8 kV.
The time constant should correspond to 0.1 ms. Electroporation leads to about
50% of cell death.
µ
11.
Quickly and carefully transfer electroporated cells into 8.5 mL of ES Medium.
Mix and distribute 1 mL of transfected cells to each feeder plate.
3.3.3. Picking and Freezing of Resistant Clones
Twenty-four hours after electroporation, medium is changed to Selection
Medium. In case a neomycin resistance cassette is used, cells can be selected
by the addition of G418 antibiotic powder to ES Medium at a concentration of
400
g/mL ( see Note 6 ). In these conditions, resistant colonies appear within
5-6 d after transfection ( see Note 7 ). In a typical experiment, enough recombi-
nant clones can be detected in about 2-300 picked colonies. Not all colonies
grow at the same rate, therefore picking and freezing steps can take 2-3 d each.
µ
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