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BURT G. FEUERSTEIN et aJ.
to 7.0 if necessary. Bring volume to 10 ml. Store as I ml aliquots at
-20°C.
- Denaturing solution: 70% formamidel2 x SSC. Combine 70 ml formamide and 10 ml20x sse. pH to 7.0 if necessary and bring up to 100 ml
with water. Store at 4°e. Prepare fresh weekly.
- PN solution: 0.1 M Na2HP04, with 0.1% Non idet P-40. Add 0.1 M
NaH 2 P0 4 H 2 0IO.I% Nonidet P-40 to 0.1 M Na2HP047H2010.1% Nonidet P-40 to bring to pH 8.0. Store up to I month at room temperature.
- Blocking buffer: I% bovine serum albumin in 4x SSe.
- DAPI mounting medium: Stock: Dissolve I mg 4',6-diamidino-2-phenylindole (DAPI) in 10 ml H 2 0. (Add several drops of methanol to
dissolve DAPI before adding water.). Store in foil wrapped tubes at
-20°C in appropriately sized aliquots. Stable for over I year.
Working solution: Add 1-2 III DAPI stock solution (0.1-0.2Ilg/ml final
concentration) to I ml phenylenediamine antifade mounting medium.
Protect from light at -20°e. Replace as solution darkens.
- SSC wash solutions should be changed for each new experiment. The
formamide solution can be reused for 3 washes or 20 slides before
changing. Wash solutions should also be changed if there is excess unhybridized probe in the previous experiment. Make certain that slides
are protected from light when using direct labeled probes.
- CoT-I DNA quality varies from lot to lot. Often preparations can contain a large amount of very small DNA fragments that are useless for
blocking repeat sequences. We have a special batch made, test it at I
mg/ml, and put a large amount on reserve so that we can use the same
lot for multiple experiments.
Procedure
Prepare
hybridization mix
1. Mix sequentially 20 III CoT-1 DNA (I Ilgfl.tl), 40 III of the labeled test
and 20 III of the labeled reference DNAs. The volumes can vary depending on the efficiency of labeling and the source of DNA. Add 1/10 volume 3 M sodium acetate (pH 5.3) and 2.5 volumes cold (-20°C) 100%
ethanol. Vortex vigorously, and centrifuge at 14,000 rpm in a microfuge at 4°C for 30 min.
BURT G. FEUERSTEIN et aJ.
to 7.0 if necessary. Bring volume to 10 ml. Store as I ml aliquots at
-20°C.
- Denaturing solution: 70% formamidel2 x SSC. Combine 70 ml formamide and 10 ml20x sse. pH to 7.0 if necessary and bring up to 100 ml
with water. Store at 4°e. Prepare fresh weekly.
- PN solution: 0.1 M Na2HP04, with 0.1% Non idet P-40. Add 0.1 M
NaH 2 P0 4 H 2 0IO.I% Nonidet P-40 to 0.1 M Na2HP047H2010.1% Nonidet P-40 to bring to pH 8.0. Store up to I month at room temperature.
- Blocking buffer: I% bovine serum albumin in 4x SSe.
- DAPI mounting medium: Stock: Dissolve I mg 4',6-diamidino-2-phenylindole (DAPI) in 10 ml H 2 0. (Add several drops of methanol to
dissolve DAPI before adding water.). Store in foil wrapped tubes at
-20°C in appropriately sized aliquots. Stable for over I year.
Working solution: Add 1-2 III DAPI stock solution (0.1-0.2Ilg/ml final
concentration) to I ml phenylenediamine antifade mounting medium.
Protect from light at -20°e. Replace as solution darkens.
- SSC wash solutions should be changed for each new experiment. The
formamide solution can be reused for 3 washes or 20 slides before
changing. Wash solutions should also be changed if there is excess unhybridized probe in the previous experiment. Make certain that slides
are protected from light when using direct labeled probes.
- CoT-I DNA quality varies from lot to lot. Often preparations can contain a large amount of very small DNA fragments that are useless for
blocking repeat sequences. We have a special batch made, test it at I
mg/ml, and put a large amount on reserve so that we can use the same
lot for multiple experiments.
Procedure
Prepare
hybridization mix
1. Mix sequentially 20 III CoT-1 DNA (I Ilgfl.tl), 40 III of the labeled test
and 20 III of the labeled reference DNAs. The volumes can vary depending on the efficiency of labeling and the source of DNA. Add 1/10 volume 3 M sodium acetate (pH 5.3) and 2.5 volumes cold (-20°C) 100%
ethanol. Vortex vigorously, and centrifuge at 14,000 rpm in a microfuge at 4°C for 30 min.