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Wheat Genetics Resource Center

Kansas State University
Department of Plant Pathology

4024 Throckmorton
1712 Claflin Road
Manhattan, KS 66506-5502
785-532-6176 (TEL)
785-532-5692 (FAX)
wgrc@k-state.edu

Wheat Genetics Resource Center
Kansas Wheat Innovation Center

1990 Kimball Ave
Manhattan, KS 66502
785-320-4383 (TEL)

 

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.