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HIROTAKA MATSUMI AND TETSU YANO
• The hybridization mixture is usually prepared immediately
prior to use but can be stored at - 20 °C for up to 6 months.
• The blocking solution contains 500llg/ml normal goat IgG,
5 % BSA, 0.3 M NaCl, lOOllg/ml salmon testis DNA, lOOllg/
ml yeast tRNA in PBS.
• The blocking solution is usually prepared immediately prior
to use, but can be stored at - 20 °C for up to 6 months.
• Typically, the concentration of probe is between 0.5 - 4llg/ml.
Increasing the probe concentration tends to result in higher
levels of background as well as in situ hybridization signal.
• The chromogen solution contains 3, 3-diaminsobenzidine
tetrahydrochloride (DAB), H20 2, nickel and cobalt; 0.5 mg/
ml DAB/0.025 % CoClz /0.02 % NiS04(NH4hS04/ 0.01 %
H20 2/O.1 M phosphate buffer (pH 7.5)
Procedure
Unless otherwise specified, all steps are performed at room temperature.
All the steps up to hybridization should be carried out in
RNase free conditions.
Solutions should be autoclaved or prepared with sterile ingredients using RNase free equipment. Equipment should be autoclaved, or baked at 240 °C for 4 hrs. (Gauses should be baked at
180°C for 2 hrs.)
Digestion of ds eDNA probe with restriction endonucleases
1. Dilute the ds eDNA to a concentration of 50 Ilg/ml with TE
buffer in a clean microcentrifuge tube.
2. Pipet the following into a clean micro centrifuge tube; ds
eDNA, lOx restriction buffer and H20
3. Add a restriction endonuclease and incubate the reaction
mixture for 10 - 30 mins at the recommended temperature
(in general, 37°C).
HIROTAKA MATSUMI AND TETSU YANO
• The hybridization mixture is usually prepared immediately
prior to use but can be stored at - 20 °C for up to 6 months.
• The blocking solution contains 500llg/ml normal goat IgG,
5 % BSA, 0.3 M NaCl, lOOllg/ml salmon testis DNA, lOOllg/
ml yeast tRNA in PBS.
• The blocking solution is usually prepared immediately prior
to use, but can be stored at - 20 °C for up to 6 months.
• Typically, the concentration of probe is between 0.5 - 4llg/ml.
Increasing the probe concentration tends to result in higher
levels of background as well as in situ hybridization signal.
• The chromogen solution contains 3, 3-diaminsobenzidine
tetrahydrochloride (DAB), H20 2, nickel and cobalt; 0.5 mg/
ml DAB/0.025 % CoClz /0.02 % NiS04(NH4hS04/ 0.01 %
H20 2/O.1 M phosphate buffer (pH 7.5)
Procedure
Unless otherwise specified, all steps are performed at room temperature.
All the steps up to hybridization should be carried out in
RNase free conditions.
Solutions should be autoclaved or prepared with sterile ingredients using RNase free equipment. Equipment should be autoclaved, or baked at 240 °C for 4 hrs. (Gauses should be baked at
180°C for 2 hrs.)
Digestion of ds eDNA probe with restriction endonucleases
1. Dilute the ds eDNA to a concentration of 50 Ilg/ml with TE
buffer in a clean microcentrifuge tube.
2. Pipet the following into a clean micro centrifuge tube; ds
eDNA, lOx restriction buffer and H20
3. Add a restriction endonuclease and incubate the reaction
mixture for 10 - 30 mins at the recommended temperature
(in general, 37°C).