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Star Republic: Guide for Biologists

Phage library screening --- Southern

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.