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Method: Transfer of DNA from Bacterial Colonies to Nylon Membrane or Nitrocellulose

April 19 1990

Jim Howe


Purpose:

Time required:

Special Materials:

Procedure:

Day 1

  1. Plate the transformation mixture onto selective medium plates and leave in hood with cover off until agar surface is dry. Incubate at 37 degrees C for 12-14 hours (smaller colonies give cleaner hybridization signals); refrigerate plates for 30-60 minutes to firm up the agar.
  2. 82 mm filters should be autoclaved in advance; first wet the filters with dH2O and place between layers of Whatman paper. Then wrap in aluminum foil and autoclave at 15 lbs/sq.in. using the liquid cycle.

Day 2

  1. Label the filters then place (with label face down) onto bacterial plate. When filter is completely wet stab agar with needle dipped in India ink in >3 places peripherally to make asymmetric markings.
  2. Prepare four pieces of Whatman 3 MM paper for colony lysis and DNA transfer; lay these in a plastic tray then saturate each with one of the following solutions:

     	a.  10% SDS 	b.  0.5 N NaOH 1.5 M NaCl 	c.  1.5 M NaCl 0.5 M Tris-HCl pH 7.4 	d.  2X SSC 

    Pour off any excess liquid for this will cause colonies to swell and blur subsequent hybridization signals.

  3. Peel the filter from the agar plate with blunt-tipped forceps and place upon the 10% SDS soaked paper colony side up. After three minutes transfer this to paper soaked in denaturing solution (b). After five minutes transfer to neutralizing paper (c). After five minutes transfer to paper soaked in (d).
  4. After five minutes of treatment with solution (d) dry the filters on dry 3 MM paper. Place filters between layers of 3 MM paper and bake in a vacuum oven for 1-2 hours. Store dry or wash in 0.1X SSC 0.5% SDS at 65 degrees C for 30 minutes and store wet in seal-a-meal bags.
  5. Perform hybridizations as per the standard protocol.

References:

Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp. 1.90-1.100.