Transfer Membranes

Protocol 9: Colony Hybridization and Plaque Lifts with Pure Nylon Neutral
Membranes
Membrane Preparation
If necessary, sterilize the membrane between two pieces of filter paper in the autoclave for 15 minutes.
09
protocol
Colony Transfer
Step 1:
Incubate cell colonies and plaques at 37°C until they are 0.5 – 1.0 mm in diameter. Alternatively, the cell colonies may be
grown after the membrane and appropriate cell media have been added to the plate. Place membranes on the plate with the
center touching first so that the membranes wet from the center out to the edge. Assure orientation by marking.
Step 2:
Replication. With forceps, lift the membrane from the surface of the plate and place it colony/plaque side up on two sheets
of dry filter paper. Carefully place a fresh membrane over the one with the colonies, and apply gentle pressure with a
replication tool or glass plate. Avoid smearing the colonies when separating the membranes. To amplify plasmids, transfer
the membrane to an agar plate with 200 – 250 µg/ml of chloramphenicol at 37°C for 10 hours.
Plaque Transfer
Step 1:
Cells should be plated with phage in soft agarose and incubated at 37°C until plaques are 0.2 mm. After incubation, chill
plate at 4°C for 15 minutes to set the agarose.
Step 2:
Place membrane on the plate in complete contact with the agarose and assure orientation by marking. Allow phage to
transfer for 5 minutes. Increase the transfer time if many transfers are to be performed.
Isolation and Immobilization
Lay membrane on plastic wrap with a pool of 0.5 N NaOH (add 1.0 M NaCl for plaques) for 10 minutes to denature the
nucleic acid. Remove circles from plastic wrap and transfer them to filter paper to absorb excess moisture for 2 minutes. If
filter paper absorbtion is incomplete, transfer the circles to fresh filter paper.
Colonies
Neutralize the membrane on plastic wrap with pools of 0.5 M Tris · HCl (pH 8.0), 0.5 M NaCl for 2 minutes and transfer
them to the filter paper to absorb excess moisture. If filter paper absorbtion is incomplete, transfer the circles to fresh filter
paper. Rinse in 2X SSC.
Plaques
Neutralize and incubate the membrane on filter paper saturated with 0.5 M Tris · HCl (pH 8.0), 1.5 M NaCl. Place the
membrane on sheets wet with 2X SSC and blot dry. Bake membranes until dry for 15 – 30 minutes at 80°C.
Hybridization
See prehybridization, hybridization, and post-hybridization procedures within the Southern and Northern Hybridization
section.
Detection
Autoradiograph on X-ray film at -70°C for 48 hours or more.
Probe Removal
See Southern and Northern Hybridizations for probe removal details.
t o c o l s
© 2008 AppliChem • Transfer Membranes 31