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 the plastic mask on top of the vacuum blotter. Choose or cut an appropriate openning.

11. Place the gel sandwich on top of the plastic mask. The membrane must be on top of the gel.

12. The gel should fit the opening on the mask. Seal around the edge of the blot with plastic strips, Saran wrap or parafilm.

13. Wet the sponge with transferring buffer (5X SSC or 20X SSC).

14. Close the vacuum blotter.

15. Connect to a vacuum and transfer for 1-4 hours.

16. 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).

17. Proceed to hybridization: