174
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
Subprotocol 3
Dot-Biot Assay to Test Labelling
Materials
Reagents • DNA dilution buffer: 0.1 mglml sheared salmon sperm DNA, 6x SSC (1x
SSC: 0.1SM NaCl, 0.01S sodium citrate, pH 7.0)
• Series of dilutions ofbiotinylated standard DNA ( commercially available
standard or previously used, well-labeled probe) in DNA dilution buffer,
e.g. 1, 3, 10, 20 pg/J..Ll standard DNA
• AP 7.5 buffer: 0.1 M Tris-HCl pH 7.5, 0.1 M NaCl and 2 mM MgC12
• AP 9.S buffer: 0.1 M Tris-HCl pH 9.5, 0.1 M NaCl and SO mM MgCh
• Blocking buffer: 3% BSA in AP 7.S buffer
• TE buffer: 10 mM Tris-HCl pH 7.S and 1 mM EDTA
• 7S mglml nitroblue tetrazolium (NBT) in 70% dimethylformamide and
SO mg/mlS-bromo-4-chloro-3-indolyl phosphate (BCIP) in dimethylformamide
Supplies • Nitrocellulose filter
Dot-blot for testing labeling
Procedure
1. Aliquot 1f . .tl ofthe dilutions of standard DNA and, in parallel, 1 J..Ll ofidentical dilutions of test DNA on a nitrocellulose filter.
2. Bake the filter at 80°C for 1 hr in a vacuum oven.
3. Wash filter with AP 7.S buffer for 1 min at room temperature.
4. Sealfilter in a plastic bag containing 10 ml ofblocking solution. Incubate
at 37°C for 30 min.
S. Open the bag at one end, remove the blocking solution and add a freshly
prepared solution of streptavidin-conjugated alkaHne phosphatase (diluted to a concentration of 1)lg/ml in AP 7.S). Re-seal the bag and incubate at 37°C for 30 min.
6. Remove the filter from the plastic bag and wash with AP 7.S buffer (twice
for S minutes at room temperature) followed by AP 9.S buffer (10 minutes at room temperature).
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
Subprotocol 3
Dot-Biot Assay to Test Labelling
Materials
Reagents • DNA dilution buffer: 0.1 mglml sheared salmon sperm DNA, 6x SSC (1x
SSC: 0.1SM NaCl, 0.01S sodium citrate, pH 7.0)
• Series of dilutions ofbiotinylated standard DNA ( commercially available
standard or previously used, well-labeled probe) in DNA dilution buffer,
e.g. 1, 3, 10, 20 pg/J..Ll standard DNA
• AP 7.5 buffer: 0.1 M Tris-HCl pH 7.5, 0.1 M NaCl and 2 mM MgC12
• AP 9.S buffer: 0.1 M Tris-HCl pH 9.5, 0.1 M NaCl and SO mM MgCh
• Blocking buffer: 3% BSA in AP 7.S buffer
• TE buffer: 10 mM Tris-HCl pH 7.S and 1 mM EDTA
• 7S mglml nitroblue tetrazolium (NBT) in 70% dimethylformamide and
SO mg/mlS-bromo-4-chloro-3-indolyl phosphate (BCIP) in dimethylformamide
Supplies • Nitrocellulose filter
Dot-blot for testing labeling
Procedure
1. Aliquot 1f . .tl ofthe dilutions of standard DNA and, in parallel, 1 J..Ll ofidentical dilutions of test DNA on a nitrocellulose filter.
2. Bake the filter at 80°C for 1 hr in a vacuum oven.
3. Wash filter with AP 7.S buffer for 1 min at room temperature.
4. Sealfilter in a plastic bag containing 10 ml ofblocking solution. Incubate
at 37°C for 30 min.
S. Open the bag at one end, remove the blocking solution and add a freshly
prepared solution of streptavidin-conjugated alkaHne phosphatase (diluted to a concentration of 1)lg/ml in AP 7.S). Re-seal the bag and incubate at 37°C for 30 min.
6. Remove the filter from the plastic bag and wash with AP 7.S buffer (twice
for S minutes at room temperature) followed by AP 9.S buffer (10 minutes at room temperature).