Biology Reference
In-Depth Information
2.
Add 500
µ
L of isopropylic alcohol and let shake overnight at room temperature.
3.
Label Eppendorf tubes correspondingly to lysed clones. Add 100
µ
L of sterile
DDW to each tube.
4.
Prepare a glass rod by flaming the tip of a Pasteur pipet. Collect with this instru-
ment the white DNA precipitate that formed on the bottom of a 24-well dish well.
Disperse the DNA in the water of the corresponding tube. Clean the glass rod in
sterile DDW and dry it with a paper towel. Repeat this step for the precipitates of
all different clones.
5.
Let DNA dissolve overnight at 56
°
C. Store genomic DNA at 4
°
C.
6.
Digest 15
µ
L of genomic DNA with the suitable restriction enzyme in 30
µ
L of a
final reaction volume containing 0.3
µ
L BSA, 3
µ
L buffer, 30-40 U enzyme.
Incubate overnight in an oven at 37
°
C.
7.
Load digested DNA on a 0.8% agarose gel in 1X TBE, 1
µ
g/mL ethidium bro-
mide. Separate for 6-8 h at 3 V/cm.
8.
Photograph gel on an UV-table together with a ruler.
9.
Blot the digested DNA onto N+ Nylon membrane in alkaline conditions.
10.
Mark the position of the wells on the nylon membrane with a pencil. Neutralize
the membrane twice in 2X SSC.
11.
Place filters in a plastic bag containing the minimum amount of Church buffer to
thoroughly wet them. Prehybridize for 1 h at 65
C. Radioactively label the probe
by random priming following the instructions provided in the kit. Hybridize fil-
ters overnight at 65
°
°
C in Church buffer containing at least 1
×
10
6
cpm/mL of
32
P-labeled probe.
12. Wash filters at 65
°
C in 250-500 mL of hot Washing Solution 1 and 2 for
30 min each.
13.
Expose with an autoradiography film until clear bands are visible.
3.3.5. Transient Cre Transfection
After homologous recombinant ES clones have been identified, the floxed
selection cassette can be removed by transient
Cre
transfection. This proce-
dure is particularly useful whenever inducible gene inactivation must be
accomplished: removal of the selection cassette leaves only the short 34 bp
loxP
sequence in the locus. Modification of the genomic sequence is thus
reduced to a minimum that leaves gene activity unaltered until a second
Cre
mediated recombination event takes place (
see
Note 9
). Whenever a floxed
neo-tk selection cassette is used, its deletion is essential because products of
the tk gene can generate sterility in male chimeras
(32)
.
1.
Thaw ES cells in one well of a 6-well microtiter dish seeded with feeder cells.
Expand the culture for few days to obtain one or more confluent 9-cm tissue-
culture dishes.
2.
Precipitate 20
g of pIC-
Cre
plasmid with 1/10 the volume of 3
M
Na acetate and
2 volumes of 100% ethanol. Leave the precipitate in 70% ethanol.
µ
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