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Method: Transfer of Phage DNA from Plaques to Nylon Membrane or Nitrocellulose
April 19 1990
To transfer phage DNA to nylon membranes so that a phage library can be screened by hybridization.
Special Materials: Procedure:
- Phage plaques should be grown until they reach 1.5 mm or greater diameter ( >8 hours to overnight; some plaques may not reach this size). It is important to have adequate spacing between plaques for two reasons: it will be easier to isolate individual plaques later and confluence of plaques may cause the top agar layer to stick to the transfer membrane destroying the plate. It is important to titer phage stock before plating to optimize the spacing between plaques.
- Chill the plates at 4 degrees C for >1 hour to render top agar firm.
- Label in advance 82 mm round filters (nylon membrane or nitrocellulose); 1 or more filters can be made from each plate. Prepare 500-1000 ml each of denaturing neutralizing and rinse solutions.
- Gently lay the 82 mm filter upon the top agar grasping it by the edges and allowing the center to touch down first. Take a needle (20-22 gauge) and dip this into India ink then jab briskly through the membrane and agar at several asymmetrically placed peripheral spots. Leave the filter in place for 30-60 seconds (if a second filter is made leave on for at least 1 minute).
- Carefully lift the filter from the agar by pulling up from one edge with forceps and peel slowly away from this spot.
- Place the filter in denaturing solution DNA side up for 1-3 minutes. If top agar sticks to filter agitate filter to remove at this step.
- Finally rinse the filter briefly in 2X SSC then place on Whatman 3MM paper to dry.
- Bake filters for 1-2 hours in a vacuum oven. Filters can be stored dry or washed at 65 degrees C for 30 minutes in 0.1X SSC 0.5% SDS and stored wet in seal-a-meal bags.
- Prehybridize and hybridize as per standard protocols.
- Denaturing solution:
1.5 M NaCl 0.5 M NaOH
1.5 M NaCl 0.5 M Tris-HCl pH 8.0 Rinse solution:
Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp. 2.109-2.111.