Kansas State University

MEMBRANE WASHING AND STRIPING PROCEDURE

Membrane washing

1. Remove bottle from hybridization oven and discard the solution into a radioactive waste container.
2. Remove membrane from bottle and place into a plastic box. Rinse membranes with 2X SSPE. Separate membrans by placing a piece of nylon mesh between each [Small Parts, Inc., CMN-300-D].
3. Preheat the wash solution to 65°C and add to the box with the membranes. Place the box in a 65°C waterbath.
4. Wash for 30 minutes with 2X SSPE, 30 minutes with 1X SSPE, and 30 minutes with 0.5X SSPE.

2X SSPE

Stock	1.0 L	2.0 L
20X SSPE	100 mL	200 mL
20% SDS	25 mL	50 mL
ddH20	875 mL	1,750 mL

1X SSPE

Stock	1.0 L	2.0 L
20X SSPE	50 mL	100 mL
20% SDS	25 mL	50 mL
ddH20	925 mL	1,850 mL

0.5X SSPE

Stock	1.0 L	2.0 L
20X SSPE	25 mL	50 mL
20% SDS	25 mL	50 mL
ddH20	950 mL	1,900 mL

1. After the last wash, place filters, DNA side up, on a piece of 3MM gel-blot paper. When the liquid has evaporated [do not dry for too long], and place membrane in a plastic sheet protector pretreated with Sigmacote [Sigma SL-2].
2. Check signal [400–800 cpm] with a Geiger counter.
3. In a darkroom, put membrane into a labeled x-ray cassette and add x-ray film. Place cassette at -80°C and expose for 4–5 days.
4. Develop film according to standard procedures.

Membrane stripping

Strip membrane by pouring a solution of 0.5M NaOH over the filters and wash for 1 hour at room temperature with gentle shaking. Rinse once with distilled water and then with 2X SSPE. Keep membrane in 2X SSPE at 4°C [refrigerator] until next use.