| |
| This very simple protocol describes a rapid method for screening a large number of DNA samples for homology to a known DNA sample. The radioactively labeled probe DNA is hybridized to filters to which the test DNAs have been spotted in a grid pattern. Both double-stranded and single-stranded DNA work. |
| |
1. Mark a grid on a piece of Whatman 3MM filter paper with the lines 1 cm to 2 cm apart, with enough intersections for all of the test samples and controls. Number each intersection and make a list relating the intersection numbers to specific test samples.
2. Using gloves, cut a piece of Nitrocellulose big enough to cover the grid with a 2 cm margin and make an orientation mark on the Nitrocellulose.
3. Lay the marked 3MM paper on a light box and over this place the Nitrocellulose filter. With the light on, the grid should be clearly visible through the Nitrocellulose.
4. Spot 2 to 10 μl (5 μl is usually sufficient) of each DNA sample onto the Nitrocellulose at the appropriate positions (see Hint #2). Allow the filters to air dry.
5. Float the Nitrocellulose filter in Denaturing Solution for 30 min at room temperature.
6. Carefully transfer and float the Nitrocellulose in Neutralizing Solution.
7. Incubate for 30 min at room temperature.
8. Transfer the Nitrocellulose onto a piece of 3MM paper and allow it to air dry.
9. Incubate the Nitrocellulose for at least 1 hr at 80°C in a vacuum oven (if the Nitrocellulose is heated in air, it may burn).
10. The Nitrocellulose is now ready for hybridization. Store at room temperature in a dark, cool place.
|
| Neutralizing Solution |
| 3 M NaCl
0.5 M Tris, pH 7.0
|
 |
| 0.1 M NaOH (CAUTION! see Hint #1) |
| 1.5 M NaCl
|
|
|
| |
Sodium Hydroxide
Tris
Sodium Chloride
|
| |
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
2. As you can imagine, this process can be miniaturized to produce a single filter containing several thousand DNA samples.
|
