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Screening colonies

Screening Colonies by Hybridization with Radiolabeled Probe

  1. Lysis of Colonies and Binding of DNA to Nitrocellulose Filters

  2. Labeling of DNA-Probe with 32P

  3. Hybridization to Nitrocellulose Filters Containing Replicas of Bacterial Colonies


1. Lysis of Colonies and Binding of DNA to Nitrocellulose Filters

Outline

Lifting colonies from agar plates onto nitrocellulose membranes, lyse cells, neutralize and bake them with the final result of having the bacterial DNA fixed to the membranes, ready for hybridization. 

Supplies & Equipment:

Reagents:

10% SDS (optional)
(dissolve 50 g SDS in 500 ml dH2O)
Denaturing solution (0.5 N NaOH, 1.5 M NaCl)
(dissolve 43.83 g NaCl in 300 ml dH2O, add 50 ml 10 N NaOH stock solution, dH2O ad 500 ml)
Neutralizing solution (1.5 M NaCl, 0.5 M Tris Cl [pH 7.4])
(100 ml dH2O, add 250 ml 1 M Tris Cl [pH 7.4] stock solution, dissolve 43.83 g NaCl, dH2O ad 500 ml)
2 x SSC
(50 ml of 20 x SSC stock solution, dH2O ad 500 ml)

Procedure:

  1. Turn on the vacuum oven.
  2. Cut 4 pieces of Whatman 3 MM paper to tray size. Saturate every tray with one of the above listed solutions. Roll out air bubbles with (e.g.) pipet. Pour off excess liquid.
  3. Label nitrocellulose filters with soft pencil and place them dry on agar plates. Mark the orientation by puncturing filters into the agar with the black ink needle (1 spot at 12:00, 2 spots at 04:00, 3 spots at 08:00). The black ink will simultaneously stain the nitrocellulose filter and the agar plate.
  4. Using blunt ended forceps, peel filters from their plates and place them, colony side up, for 3 minutes on the SDS-impregnated 3MM paper.
  5. Transfer for 5 minutes onto denaturing solution, using the edge of the first tray to remove excess liquid.
  6. Transfer for 5 minutes onto neutralizing solution.
  7. Transfer for 5 minutes onto 2 x SSC.
  8. Transfer onto a dry sheet of 3MM paper, let air dry for at least 30 minutes at room temperature.
  9. Sandwich the filters between sheets of 3MM paper. Fix the DNA to the filters by baking for 1 -2 hours at 80C in a vacuum oven.
  10. Any filters not used immediatley in hybridization reaction should be wrapped loosely in aluminum foil and stored at room temperature.

Time Required: 2 - 3 hours

2. Labeling of DNA-Probe with 32P

Outline:

Using DNA polymerase, templates, and primers in combination with radiolabeled nucleotide (either [a-32P]dATP or [a-32P]dCTP), a radioactive probe is synthesized. The procedure works with either exact size templates and any (random or specific) primers or with templates within a vector and specific primers.

Supplies & Equipment:

  • Prime It II Random Primer Labeling Kit (Stratagene #300385)
  • Waterbath 37C
  • Waterbath 100C
  • Microfuge
  • Reagents:

  • Random 9-mer primers or specific primers from PCR (10 M) or sequencing reaction (10 ng/l)
  • 5 x *dATP buffer (for use with [a-32P]dATP) containing dGTP, dCTP, dTTP, or 5 x *dCTP buffer (for use with [a-32P]dCTP) containing dGTP, dATP, dTTP
  • [a-32P]dATP) or [a-32P]dCTP) - not provided by the kit
  • Exo(-) Klenow fragment of DNA polymerase I (5 U/l)
  • Stop mix (0.5M EDTA, pH 8.0)
  • Procedure:

    1. Add to the bottom of a microfuge tube:
    2. Boil reaction tube in water bath for 5 minutes, then centrifuge briefly at room temperature to collect condensed liquid from cap of tube.
    3. Add the following reagents to the reaction tube:
    4. Mix the reaction components thoroughly with pipet tip.
    5. Incubate the reaction at 37 - 40C for 2 - 10 minutes.
    6. Stop the reaction by adding 2 l of stop mix.
    7. Purify probe with either one of the following kits:
    8. Count radioactivity of probe. Take 1 l probe, dilute with appropriate amount of scintillation fluid, use same amount of scintillation fluid as negative control. Count. You should have ~106 cpm per ml hybridization reaction (~ 15 million cpm per hyb).
    9. Boil probe 5 min. immediately before use.

    Time Required: 30 minutes 

    3. Hybridization to Nitrocellulose Filters Containing Replicas of Bacterial Colonies

    Outline

    After the DNA has been fixed to the filters by baking, the following steps take place in hybridization bottles and hybridization oven. Following a wash step, bacterial debris is wiped mechanically off the filter surface. Filters then are prehybridized to block binding sites, and the radiolabeled probe added thereafter will compete for specific binding sites. After washing, the air died filters are exposed to an X-ray film which shows positive colonies..

    Supplies & Equipment:

    Reagents:

    Procedure:

    1. Float the baked filters on the surface of a tray of 2 x SSC until they have become thoroughly wetted from beneath. Submerge the filters for 5 minutes.
    2. Sandwich the filters between pieces of nylon mesh (up to 8 per bottle)
      Roll stack of mesh and filters in the way shown in the picture. Add 50 ml prewash solution per bottle. Place bottle with lid on right side into hybridization oven. Prewash at 50C for 30 minutes.
    3. Using Kimwipes, gently wipe bacterial debris from the surface of the filters. Rinse filters in 2 x SSC and wash nylon mesh and bottles with water to remove SDS. Sandwich again and place in bottles as decriebed above.
    4. Add 15 ml prewarmed prehybridization solution per bottle (42C with formamide, 68C without formamide). Prehybridize for 1 - 2 hours.
    5. Boil probe for 5 minutes. Make sure the cap of the tube is secured to avoid radioactive spill when tube pops open due to heat expansion! Spin down down shortly to condensated liquid from cap. Add probe to hybridization bottle.
    6. Hybridize overnight at either 42C or 68C.
    7. When the hybridization is completed, pour off the hybridization solution and add 50 ml of wash solution 1. Wash for 15 minutes at 68C (42C also possible).
    8. Wash two times with 50 ml of wash solution 2 at 68C for 1 - 1.5 hours.
    9. optional: Wash once in wash solution 3 at 68C for 1 hour. Only necessary if high background demands washing at high stringency.
    10. Dry the filters on paper towels at room temperature. Fix filters (colony side up) with tape on film cassette-size piece of filter paper. Help later orientation with fluorescent marker pen. Cover with Saran Wrap. Expose in cassette at -80C for 2 hours. Expose again longer if needed.

    Time Required: 4 hours + overnight hybridization + 4 hours