John M. S. Bartlett.pdf - Bio-Nica.info

Digoxigenin 351
to mix, and denature samples for at least 2 min at 85°C using a heating block or
thermal cycler.
2. Transfer 6 µL of each sample to the wells of the sequencing gel. Run gel at 35 mA for 2 to
4 h (until the upper dye front has reached the bottom end of the gel).
3. Remove glass plates from electrophoresis apparatus. Carefully take off the glass plate
covered with Sigmacote on the inner side using a scalpel blade as a lever. Cover gel with
nylon membrane (avoid air bubbles!) and cover with one layer of Whatman filter paper.
Put on second glass plate and add approx 2 kg of weight. Leave for 30 min (see Note 7).
Protect nylon membrane with one layer of plastic foil.
4. Crosslink nylon membrane (plastic wrapped, blotted side down) on an ultraviolet light box
at 302 nm for 3 min (see Note 8). The membrane may now be air-dried, sealed in a plastic
bag (see Note 9), and stored at 4°C until further use.
3.4.2. Visualization of Sequencing Bands
Visualization of sequencing results is performed in plastic hybridization bags (one
blot per bag) at room temperature using the Digoxigenin Detection Kit (Boehringer).
After crosslinking, wet blots may be used immediately for immunological detection.
The following recipe applies to a 4∞4 lane gel (gently agitate membrane, except during
the final step when incubating with substrate solution).
1. Incubate nylon membrane (in hybridization bag) for at least 30 min in approx 30 mL of
blocking solution (0.5 g/100 mL TBS; see Note 10).
2. Wash blot three times in approx 100 mL of TBS (10 min each).
3. Incubate membrane with 50 mL of alkaline phosphatase-conjugated antidigoxigenin
antibody for 1 h (50 µL antiserum/50 mL of TBS).
4. Wash three times in TBS (20 min each).
5. Prepare 30 mL of substrate solution (mix immediately before use): 150 µL of NBT
(77 mg/mL in 70% N,N-dimethyl formamide [v/v]); 112 µL X-phosphate (50 mg/mL in
N,N-dimethyl formamide); and 30 mL of alkaline phosphatase buffer, pH 9.5.
Thoroughly remove washing solution from membrane. Add substrate solution. During
this incubation step, cover plastic bag with black light protector and do not agitate (see
Note 11).
6. Stop developing process when faint sequencing bands are beginning to appear. Add approx
1 L of deionized water to hybridization bag. Cut bag open and carefully remove membrane.
Allow membrane to air-dry on sheets of Whatman filter paper or paper towels. Store
stained, dried membranes in the dark (see Note 12). An example of a stained membrane
is shown in Fig. 2.
4. Notes
1. Molecular biology-grade reagents are used for all experiments.
2. Oligonucleotide primers for PCR and sequencing may be designed by hand, but use of a
computer program, such as Oligo (National Biosciences, Oslo, Norway), greatly facilitates
this task. We have had good experience with primers ranging from 18 to 25 bp in length
for both PCR and sequencing. The sequencing primer may be internally nested or identical
in position and/or length to the primer used for PCR. Primers used in our laboratory were
synthesized on an Applied Biosystems 394 DNA synthesizer at the Genzentrum of the
University of Munich. Sequencing primers were custom-made and hapten-labeled using
digoxigenin-3-O-methylcarbonylε-amino-caproic acid-N-hydroxy-succinimide ester and
a 5′-oligopeptide linker (#1333054, Boehringer Mannheim).