Southern blotting onto a nylon membrane with alkaline transfer

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Note: with a positively charged nylon membrane, the transferred DNA becomes covalently linked to the membrane if an alkaline transfer buffer is used.

Materials required
0.4 M (for charged membrane) or 0.25 M (for uncharged membrane) NaOH
Positively charged or uncharged nylon membrane ("Amersham" Hybond-N (uncharged) or Hybond-N+ (positively charged) nylon; "Bio-Rad" Zeta-Probe positively charged nylon; "Ambion" BrightStar-Plus positively charged nylon)
2xSSC

  1. Run a gel, cut and measure it and take a photo. Prepare membrane, pieces of Whatman 3MM and paper towels. Work in gloves and handle the membrane with forceps.
  2. Assemble the transfer rack as shown on the scheme. Fill the dish with 0.4M NaOH. Place the gel on support face down, place pieces of Parafilm along the edges of the gel, pour some 0.4M NaOH on the top of the gel and cover with a nylon membrane and 4-5 pieces of Whatman 3MM. Avoid air bubbles in between the gel and nylon membrane and Whatman 3MM! Any trapped air should be carefully removed by rolling a 10-ml glass pipet over the surface.
  3. Cover Whatman with a stack of paper towels. Lay a glass plate on top of the stack and place a 0.2-0.4-kg weight on top. Leave overnight.
  4. Recover membrane and cut one corner to mark the orientation. Rinse the membrane in 2xSSC, then place it on a sheet of Whatman 3MM paper and allow to dry. Baking or UV cross-Iinking is optional with positively charged membranes; however, DNA on uncharged membranes should be cross-linked by UV exposure. Store membranes at room temperature between pieces of Whatman 3MM wrapped in foil.

 

                 

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