2. Prepare the Whatman 3MM filter paper.
Cut two 9- × 18-cm rectangles of Whatman 3MM paper to serve as wicks. Cut two 7.5- × 10-cm
rectangles for the transfer stack (see diagram at right).
3. Prepare the paper towels.
Cut enough 7.5- × 10-cm rectangles of brown paper towels to form a 2-inch stack when compressed.
D. Set up the transfer stack (see Figure 2)
Note: Always wear gloves when handling the membrane and wicks;otherwise, oil and grease from your fingers will interfere with the transfer and subsequent hybridization.
1. Place 250 mL of 10× SSC in a shallow container measuring approximately 11 × 20 cm.
2. Wet the two paper wicks in the 10× SSC. Leave them in the 10× SSC until you perform Step 4.
3. Place an inverted gel casting tray or other support (see Figure 2) in the shallow dish. The 10× SSC should not cover the support.
4. Lay the two wicks (one on top of the other) over the inverted casting tray, so that the ends of both wicks are well submerged in the 10× SSC.
5. Make sure that no air bubbles are trapped between the wicks. “Rolling” over them with a pencil or plastic pipet can help squeeze out air bubbles.
6. Remove the gel from the neutralization buffer. Lay it, with the open side of the wells facing down, on the wicks (on top of the inverted casting tray). Cut off a small piece of one lower corner of the gel and record which corner you cut in relation to the position of the DNA on the gel. Make sure no air bubbles are trapped between the gel and the wicks. Gently roll a pencil or pipet over the gel to eliminate bubbles.
7. Remove the membrane from the 10× SSC in the weigh boat and carefully lay it on the gel, with the side you wrote on contacting the gel. The membrane is narrower than an 8- × 10-cm gel, so be sure that the lanes of the gel that contain DNA are covered. Trim off the exposed portion of the gel, carefully avoiding the wick.
8. Cut off a small lower corner of the membrane to match the cut lower corner of the gel. (This will help you orient the membrane properly after hybridization.)
9. Lay strips of plastic wrap or Parafilm® around the gel so that the rest of the wick area on the casting tray is covered, if it is not already. If your gel has areas not covered by the membrane, cover them too, but do not cover the membrane. This ensures that the transfer buffer’s only migration path is through the gel and membrane (i.e., it prevents the edge of a paper towel or other stack components from accidentally contacting the wick or uncovered portions of the gel).
10. Place the two dry 7.5- × 10-cm rectangles of Whatman 3MM paper neatly on top of the membrane.
11. Place the stack of dry, cut paper towels neatly on top of the Whatman 3MM paper.
12. On top of the paper towels, place a flat piece of plastic or glass. On top of this, for weight (~500 g), place a 400-mL beaker full of water. 13. Allow the transfer stack to sit overnight.
E. Take down the stack, rinse the membrane, and bake the gel
The lower part of the paper towel stack should be completely saturated with buffer, but the paper towels themselves should not be in contact with the wicks or with the buffer in the reservoir. There should still be buffer in the reservoir. The wick ends should still be submerged.
1. Place 100 mL of 2× SSC into a plastic container with a tightfitting lid. You will use this in Step 5.
2. Remove the 400-mL beaker weight from the transfer stack. Discard the paper towels to expose the Whatman 3MM paper sheets.
3. With gloved hands, carefully leaving what remains of the stack together as a unit, turn the gel, membrane, and Whatman 3MM paper over. With a soft lead pencil, pierce the wells of the gel to mark the locations of the wells on the membrane (Figure 3).
4. Peel the gel off the membrane and discard it. If you used CarolinaBLU™ or methylene blue to stain the gel, the high-molecular-weight DNA bands will still be visible: this is normal, and does not mean that transfer failed to occur.
5. Peel the nylon membrane away from the two sheets of 3MM paper and place it in the 2× SCC prepared in Step 1. Gently agitate the container for 30 min. Note: The membrane should not bend or crinkle. Do not reduce the wash time.
6. With gloved hands or with blunt forceps, place the nylon membrane on a piece of Whatman 3MM filter paper. Allow it to air dry for at least 5 min. Then, write your group name on a second piece of Whatman paper, place it over the membrane, and then tape the pieces of Whatman paper together, avoiding the membrane.
上一篇：SOUTHERN BLOTTING 下一篇：Genomic Southern Blot 共7页: 上一页 [1] [2] [3] [4] 5 [6] [7] 下一页 |
