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Colony Hybridization

Method

Allow plates to grow O/N. Fairly large sized colonies, not very dense with no satellites, are easiest for picking.

  1. Lift colonies onto a dry nylon or nitrocellulose filter. Allow to lie in place until filter is completely wetted, usually only 20 seconds or so.
  2. Mark the filter and plate with an 18g needle three times around edge, not evenly spaced for orientation. Lift filter off plate carefully with a forceps. Entire colonies should be lifted with filter. Let plate sit 6 hours at 37°C, or O/N at RT to regrow colonies.

    For next steps have prewetted Whatman paper already set-up.

  3. Place filter colony side up on Whatman paper prewetted with Solution A for 3 minutes.
  4. Blot underside of filter on paper towel and repeat step 3.
  5. Blot underside of filter on paper towel. Place filter on Whatman prewetted in Solution B for 5 minutes.
  6. Blot underside of filter on paper towel. Place filter on Whatman prewetted in Solution C for 3 minutes.
  7. Blot underside of filter on paper towel. Place filter on Whatman prewetted in 2X SSC for 30 seconds.
  8. Blot underside of filter on paper towel. Dry filters in oven at 80°C for 30 minutes or until dry. Alternatively, dry under a heating lamp.
  9. Crosslink membranes in UV crosslinker.
  10. Prehybridize in Rapid Hyb™ Buffer if using nylon filters or in prehyb solution below if using nitrocellulose, 65°C for 10 minutes to 1 hour.
  11. Hybridize in Rapid Hyb™ Buffer if using nylon filters or in hyb solution below if using nitrocellulose, 65°C for O/N. Use 2 X 105 cpm/ml probe.
  12. Wash 2X at RT, 10 minutes, and 2X at 65°C, 30 minutes each, in Wash Buffer I.
  13. Wash 1X at 65°C, 20 minutes, in 0.2X SSC/0.2% SDS.

    Monitor all washes with Geiger counter!!

  14. Wrap damp blots in plastic wrap and expose to film, no longer than 2 hours at RT.
  15. Using pinpricks to match filter to film to plate, pick positive colonies for minipreps.

Solutions

Solution A

  • 10% SDS

Solution B

  • 0.5M NaOH
  • 1.5M NaCl

Solution C

  • 0.5M Tris, pH 8
  • 1.5M NaCl

Prehyb (Nitrocellulose Membranes)

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  • 0.4 g BSA
  • 0.08 ml 0.5M EDTA
  • 21.0 ml NaPi pH 7.2
  • 14 ml 20% SDS
  • 4.9 ml H2O

Hyb (Nitrocellulose Membranes)

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  • 0.4 g BSA
  • 0.08 ml 0.5M EDTA
  • 21.0 ml NaPi, pH 7.2
  • 2 ml 20% SDS
  • 17 ml H2O

Wash Buffer I

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  • 182 ml H2O
  • 8 ml 1M NaPi
  • 0.4 ml 0.5M EDTA
  • 10 ml 20% SDS

NaPi for 1 L

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  • 134 g Na2HPO4
  • 4 ml H3PO4
  • Check pH


Notes

  1. Always have a negative control, i.e., empty vector ligation to determine hybridization background.
  2. After lifts, never allow plates to overgrow. Satellite colonies will always form around a clone and can confuse results. O/N at RT works best for regrowing.
  3. There is no need to overwet Whatman papers during lysis steps.
  4. Any normal Southern blot protocol will work. Remember, when using nitrocellulose filters, to not use formamide-based solutions, since the filters will become brittle and fall apart.
  5. Positive colonies should be quite obvious after 1- to 2-hour exposure. If it takes longer to see a signal, you likely do not have any inserts on your plate.

*Adapted from protocols originating in the laboratories of Dr. Sharon Dent and Dr. William Klein at MD Anderson Cancer Center

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© 2013 The University of Texas MD Anderson Cancer Center