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Wheat Genetics Resource Center

Kansas State University
Department of Plant Pathology

4024 Throckmorton
1712 Claflin Road
Manhattan, KS 66506-5502
785-532-6176 (TEL)
785-532-5692 (FAX)
wgrc@k-state.edu

Wheat Genetics Resource Center
Kansas Wheat Innovation Center

1990 Kimball Ave
Manhattan, KS 66502
785-320-4383 (TEL)

 

PREHYBRIDIZATION AND PROBE LABELING

Prehybridization

    1. Set hybridization oven to 65°C.
    2. Thaw 32P isotope in hood.
    3. Place membrane in hybridization tube.
    4. Add 50 mL of prehybridization buffer to each tube.
Prehybridization buffer [for 4 probes] 
ddH20
125 mL
20X SSPE
60 mL
100X Denhardt's solution
10 mL
20% SDS
5 mL

Salmon sperm DNA [10 mg/mL; boil for 5 minutes and set on ice 5 minutes before adding]

1 mL
TOTAL
200 mL
  1. Prehybridize membranes for at least 6 hours to overnight.

Probe labeling 

    1. Heat water to boiling in a beaker on a hot plate.
    2. Labeling components are
Labeling components
DNA
1.0 µL [20–50 ng]
ddH20
4.0 µL [total volume of H20 and probe = 5 µL]
Oligo primers
1.0 µL
dNTPs [5 mM]
1.5µL

Klenow polymerase

1.0 µL
10X Klenow buffer
1.5µL
dCTP 32P
5.0 µL
TOTAL
15.0 µL
  1. Conbine ddH20, oligo primer, and DNA in a 1.5-ml microfuge tube, boil for 4 minutes, and place on ice.
  2. Add dNTPs, 10X buffer, Klenow enzyme, and 32P. Mix, spin briefly, and allow reaction to go overnight at room temperature or place at 37°C for 2 hours.

Column preparation

  1. Add glass wool to the base of a 1-mL syringe and place in a 15-mL centrifuge tube.
  2. Using a transfer pipet, fill the syringe with a solution of Sephadex G-50 in TE. Avoid getting air bubbles in the column.
  3. Centrifuge tubes for ~30 seconds, refill tubes with fresh Sephadex G-50 solution, and centrifuge at 1,500 RPM for 4 minutes.
  4. Add 200 µL of TE to each column and centrifuge for 4 minutes at 1,500 RPM.

Probe purification

  1. Add 185 µL of TE to each probe.
  2. Cut the cap off the tube and withdraw all of the probe solution from the tube with a pipet. Place an empty tube under the syringe and add the probe to the top of the column.
  3. Spin the column for 4 minutes at 1,500 RPM.
  4. Discard the column; remove tube with pruified probe and recap.
  5. Add a cap-lock to the tube, boil for 4 minutes, and place on ice.