DIG Hybridization System Protocol
(I) DNA Probe (PCR Probe) preparation
10 x Reaction Mix
5 x dNTPs 1 mM dATP 7.1 μl of 10 mM
1 mM dCTP 7.1 μl of 10 mM
1 mM dGTP 7.1 μl of 10 mM
0.65 mM dTTP 4.6 μl of 10 mM
0.35 mM Dig-dUTP 25 μl of 1 mM
20.5 μl of H2O
71.4 μl total volume
PCR Reaction
Primers
10 x Reaction Buf. 5 μl
5 x dNTPs 10 μl
Template
Enzyme 2 μl
H2O
50 μl
30 cycle should be done.
After PCR reaction, probe should be purified by Qiaquick PCR Kit.
(II) Hybridization
Hybridization solution
0.25 M Na2HPO4 pH. 7.2 ([Na+]=0.5)
7 % SDS
0.1 % SDSa
1 mM EDTA
1 % BSA
Hybridizaton Solution with PEG and Formamide
10% PEG 6000 50 g
7% SDS 35 g
0.25M NaHPO4 125 ml of 1 M stock
0.25 M NaCl 25 ml of 5M stock
50% Formamide 250 ml
1 mM EDTA1 1 ml of 0.5 M Stock
Yeast total RNA 1 ml of 100mg/ml stock
10 mM DTT 0.77 g
DW
Total 500 ml
0.5M Na2HPO4 ([Na+]=1) Stock solution
Na2HPO4 142g
リン酸 8ml
Total 2l
Probe (1-50 ng/ml) should be denatured by boiling for 10 min, not by alkali treatment.
Hybridization should be done at least for 15 hours at 65°C. Probe は250-10000倍希釈
Washing solution 1 and 2
1 % SDS
500 mM Na2HPO4 pH. 7.2
1 % SDS
50 mM Na2HPO4 pH. 7.2
Each washing should be done two times for 30 min at 65°C.
TTBS (Tris buffered saline)
500 mM NaCl, 0.1% Tween20
50 mM Tris-HCl, pH. 7.5
After washing, membrane is washed by TTBS for several times to remove SDS.
(III) Detection
1. Block for 30 min in blocking solution at RT.
0.5 % Casein-TTBS Blocking Solution used in the in situ system
2. Antibody reaction
A tube including the antibody is centrifuged at maximum speed for 5 min before use.
Antibody solution
1/10,000 diluted anti-Dig antibody AP conjugated in Blocking solution
Incubate membrane for 30 min at RT in antibody (1/10,000) solution.
Wash three times in TTBS for 15 min.
3. Chemiluminescence detection
Wash two times in Buffer 3 for 5 min at RT.
After that, membrane is applied in Lumigen PPD solution.
Incubate for 15 min at 37° C and expose to X-ray film first time for 30 min.
Buffer 3.
100 mM Tris-HCl pH 9.5
100 mM NaCl
50 mM MgCl2
Lumigen PPD solution:
1/100 diluted Lumigen PPD in buffer 3.
(IV) Dehybridization
1. Rinse the membranes three times in water.
2. Incubate in NaOH, 0.2 M; SDS, 0.1 % at 37°C for 30 min.
3. Rinse them in TBS.
4. The membrane could be dried and stored.
Dehybridization Solution
0.2 M NaOH
0.1 % SDS
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