1. Run agarose gel with markers.
2. Stain the gel and photograph with a ruler along the side of the markers.
3. Denature in 1.5 M NaCl, 0.25 M NaOH for 30 minutes at room temperature with gentle shaking.
4. Neutralize in 0.5 M Tris-HCl pH 7.5, 1.5 M NaCl, 0.1 M EDTA for 30 minutes at room temperature with gentle shaking.
5. Cut membrane to slightly bigger than the gel. Cut six pieces of Whatman 3MM paper to
the same size.
(optional) Mark the position of wells with a pen/pencil on the membrane.
6. Prewet the membrane and 3MM papers in the transferring buffer (5X SSC or 20X SSC).
7. Place the gel inverted on top of 3 Whatman 3MM paper. Avoid trapping
any bubbles between the gel and the 3MM paper.
8. Place the membrane on top of the gel. Make sure no bubbles are
trapped between the gel and the membrane.
(optional) Align the wells and marker positions on the membrane.
9. Place 3 Whatman 3MM paper on top of the membrane. Avoid trapping
any bubbles between the membrane and the 3MM paper.
10. Place a heavy object with a flat surface into a transferring tray (e.g.,
a rack or heat block. Place a plastic/glass plate on top if necessary.)
11. Place a few paper towels on top of the flat surface. Allow them
to extend beyond the surface to touch the bottom of the tray.
13. Place the gel sandwich on top of the paper towels. The membrane
must be on top of the gel.
14. Seal around the edge of the blot with Saran wrap or parafilm.
15. Place a pile of paper towels on top of the 3MM paper.
16. Place a plastic/glass plate on top.
17. Weight it down with 500 g weight (e.g., Maniatis).
18. Fill the transferring tank with 5X SSC or 20X SSC.
19. Transfer for 6-18 hours. Change paper towels if necessary.
20. Air-dry. UV cross-link (e.g., UV Crosslinker or hand held
UV light 2-5 minutes) or bake at 80 °C in a vacuum oven for 2 hours
(nitrocellulose filter only).
21. Proceed to hybridization:
Southern --- Chemiluminescence detection
Southern --- radioactive probe