26 FISH on Insect Cells Transfected with Heterologous DNA
325
Labeling of the probes
1. The plasmid probe which has been used to transfect the Schneider cells
is now used as the specific FISH probe (e.g. a derivative of the plasmid
pRmHa-3 (Bunch et al. 1988». Thus, it has to be labeled, e.g. with biotin by nick translation using the kit from Boehringer Mannheim (Cat.
No. 1745816).
2. For this purpose 1 ~g of the probe is diluted in 16 ~l of double distilled
water, and 4 ~l of the nick translation solution is added. This solution is
mixed carefully with a tip of a 20 ~l Eppendorf pipette and the 1.5-~1
microtube is incubated at 15°C for 90 min.
3. During this time, a Sephadex column can be prepared. First, a 1-ml
syringe is filled up to the O.I-mlline with silanized glass wool.
4. Then, Sephadex G80 solution is filled up to the 1.0-mlline (Sephadex
G80 solution: 8 g of Sephadex G80 powder is dissolved in 100 mIl x TE
solution and incubated overnight at RT; after that it is ready for use
and can be stored at RT for several months).
5. Put the syringe in a 15-ml tube and put it in a centrifuge at 3000 rpm for
1 min to separate the TE solution from the Sephadex.
6. Repeat steps 4 and 5.
7. Add 100 ~11 xTE solution on the prepared Sephadex column and repeat step 5 to wash the column.
8. Now the Sephadex column is ready for use and can be stored for 2-3 h
at 4°C before use. Do not forget to discard the 1xTE solution collected
in the 15-ml tube.
9. Add 20 ~l of "stopmix" and 60 ~l of 1xTE to the nick translation solution from step 2 ("Stopmix": 0.25% bromphenol blue, 0.25% dextran
blue, 0.5 M EDTA).
10. The 100 ~l solution from step 9 is put onto the sephadex column from
step 8 and centrifuged at 3000 rpm for 3 min. The light blue solution,
(due to the dextran blue of the "stopmix"), coming out of the column,
is collected in the 15-ml tube. Usually, the resulting solution is approx.
100 ~l. As 1 ~g has been introduced into the nicktranslation reaction,
the DNA concentration of the solution is around 10 ng/~l.
325
Labeling of the probes
1. The plasmid probe which has been used to transfect the Schneider cells
is now used as the specific FISH probe (e.g. a derivative of the plasmid
pRmHa-3 (Bunch et al. 1988». Thus, it has to be labeled, e.g. with biotin by nick translation using the kit from Boehringer Mannheim (Cat.
No. 1745816).
2. For this purpose 1 ~g of the probe is diluted in 16 ~l of double distilled
water, and 4 ~l of the nick translation solution is added. This solution is
mixed carefully with a tip of a 20 ~l Eppendorf pipette and the 1.5-~1
microtube is incubated at 15°C for 90 min.
3. During this time, a Sephadex column can be prepared. First, a 1-ml
syringe is filled up to the O.I-mlline with silanized glass wool.
4. Then, Sephadex G80 solution is filled up to the 1.0-mlline (Sephadex
G80 solution: 8 g of Sephadex G80 powder is dissolved in 100 mIl x TE
solution and incubated overnight at RT; after that it is ready for use
and can be stored at RT for several months).
5. Put the syringe in a 15-ml tube and put it in a centrifuge at 3000 rpm for
1 min to separate the TE solution from the Sephadex.
6. Repeat steps 4 and 5.
7. Add 100 ~11 xTE solution on the prepared Sephadex column and repeat step 5 to wash the column.
8. Now the Sephadex column is ready for use and can be stored for 2-3 h
at 4°C before use. Do not forget to discard the 1xTE solution collected
in the 15-ml tube.
9. Add 20 ~l of "stopmix" and 60 ~l of 1xTE to the nick translation solution from step 2 ("Stopmix": 0.25% bromphenol blue, 0.25% dextran
blue, 0.5 M EDTA).
10. The 100 ~l solution from step 9 is put onto the sephadex column from
step 8 and centrifuged at 3000 rpm for 3 min. The light blue solution,
(due to the dextran blue of the "stopmix"), coming out of the column,
is collected in the 15-ml tube. Usually, the resulting solution is approx.
100 ~l. As 1 ~g has been introduced into the nicktranslation reaction,
the DNA concentration of the solution is around 10 ng/~l.