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Plating library
1. Use 6.5 ml top agarose per 150 mm plate.
Dilute phages to give 15,000 - 20,000 cfu/150 mm plate. If higher
phage number is used, shorter incubation time may be needed.
2. Plate phage library as described in
Phage library --- tittering without the serial dilution.
3. Incubate at 37 °C for overnight.
Lifting of phage clones
4. Cool plates at 4 °C for > 1 hour.
5. Label nitrocellulose filters with pencil.
6. Holding filter at edge with blunt ended forceps,
place filter onto plate surface. Let the wetted area spread
to edges of filter.
7. Mark filter in 3 asymmetric locations by stabbing
through the filter and agar with 18 gauge needle connected
to a syringe filled with Indian black ink. Let the filter sit
on the plate for 1 minute.
8. Using blunt ended forceps, gently lift the filter off the
plate.
9. Repeat lifting for the duplicate filter. Let the filter sit
on the plate for 2 minutes. More than 2 duplicates can be made
from each plate.
Denaturing
10. Place filter (DNA side up) for 1-5 minutes in a tray
containing 0.5 M NaOH, 1.5 M NaCl.
11. Neutralize for 5 minutes in 0.5 M Tris-HCl pH7.4, 1.5 M NaCl.
12. Rinse twice in 2X SSC.
13. Place filter on 3MM Whatman paper (DNA side up) to dry.
14. Sandwich each filter between 3MM Whatman paper and
bake in a vacuum oven at 80 °C for 2 hours. UV crosslink if using
a nylon membrane.
Hybridization
15. Probe labeling and hybridization is as described in the following
protocols.
Southern --- Chemiluminescence detection
Southern --- radioactive probe
Secondary Screen
16. Align the plate with the autoradiogram to locate positive plaques.
17. Pick plagues with sterile pasture pipette by stabbing through the
positive plaques.
18. Place pipettes in 1 ml of 10 mM Tris-HCl pH7.5, 10 mM MgCl2, 50 mM NaCl.
19. Let stand at room temperature for 1-2 hours or 4 °C for 4 hours to overnight.
20. Dilute an aliquot 1:100 in 10 mM Tris-HCl pH7.5, 10 mM MgCl2, 50 mM NaCl.
21. Plate 1-20 ml of the diluted phage clone as in step 2.
22. Repeat screening.
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