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

266 Case-Green, Pritchard, and Southern
3. Incubate at 25°C for 1 h.
4. Incubate at 80°C for 5 min.
5. If desired, purification can be performed using a commercially available purification kit.
3.3. Hybridization (see Note 4)
3.3.1. Hybridization Assay (see Notes 5 and 6)
1. Make up a solution of the target (50–100 fmol, see Subheading 3.2.1.) in 100 µL of
buffer.
2. Soak a tissue in 1X buffer and place around the inner perimeter of a Petri dish to create
a 100% humidity chamber.
3. Place the solution as a puddle in the middle of the Petri dish and place a strip of array
(30 × 2 mm) on top.
4. Incubate for 1hr at the required temperature.
5. Wash the array in fresh buffer solution and blot dry.
3.3.2. Hybridization Reaction for Polymerase Assay (see Note 7)
This method is the same as used in Subheading 3.3.1. except that a solution of
1 to 5 pmol of single-stranded DNA target (see Subheading 3.3.2.) in a 1 M NaCl
solution is made in step 1.
3.3.3. Hybridization for Ligation Assay (see Note 8)
This method is the same as used in Subheading 3.3.1. except that a solution of
1 to 5 pmol of single-stranded DNA target (see Subheading 3.3.2.) and 5 to 10 pmol of
reporter oligonucleotide in a 1 M NaCl solution is made in step 1.
3.4. Enzyme-Catalyzed Assays (see Notes 9–11)
3.4.1. Ligase Assay
1. Make up a solution of DNA ligase (1 U/µL) in 1µ ligase buffer (100 µL) and place as a
puddle in a converted Petri dish.
2. Place a strip of array (30 × 2 mm) that has undergone hybridization according to Subheading
3.3.3. face down in the puddle, ensuring that the surface is completely covered by
the solution.
3. Incubate at 37°C (or 65°C for STR arrays) for 1 to 8 h.
4. Heat in 50/50 water/formamide v/v solution at 100°C for 5 min.
5. Blot dry and image.
3.4.2. Polymerase Assay (see Note 12)
1. Make up 40 µL of a solution containing sequenase (0.5 U/µL), DTT (2.5 mM), polymerase
buffer (1µ), and the appropriate ddNTP (25 nCi/µL).
2. Soak a tissue in water and place round the inside of a Petri dish.
3. Add the solution as a puddle to the middle of the Petri dish.
4. Float a strip of prehybridized array (see Subheading 3.3.2., 1 × 30m) face down on
the solution.
5. Incubate at 37°C for 1 h.
6. Wash away unbound material with Tris-EDTA (TE) buffer at 65°C for 5 min.
7. Blot dry and image.