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Protocol: RG57 genomic DNA fingerprinting

Radioactive random-primer labeling Southern hybridization protocol

  1. Production of a 32P-labeled probe.
    1. Thaw all labeling components except for the Klenow Fragment, and place on ice. Keep the Klenow at -20° C until just before use. We use the BMB random primer labeling kit.
    2. Place 50 ng RG57 template DNA in a clean screw cap microfuge tube and dilute with water to a final volume of 14 ul. Put the tube in a 95-100° C heat block for 3-5 min, then quickly place the tube on ice for 5 min.
    3. Add the following to the microfuge tube containing 14 ul denatured DNA:
Random primers (from kit) 2.5 ul
Random primer buffer mix (from kit) 2.5 ul
Add the following behind a shield  
32P-labeled dCTP 5.0 ul
Klenow fragment 1.0 ul  Add the Klenow fragment last
Total volume 25 ul
Incubate for about 1 hour at 37° C.  
  1. Purification of 32P-labeled probes.
    1. For each probe, use 1 Bio-Rad micro bio-spin column.
    2. Shake the column several times to resuspend the gel and to remove bubbles.
    3. Snap off the tip and place the column in a 2.0 ml microfuge tube. Remove the cap from the column. Let the column drip for about 2 min. and discard the drained buffer.
    4. Centrifuge for 1.5 min at 7,000 in the small microfuge. Discard the drained buffer.

      5. Do all of the following steps behind a shield

    5. Add 50 ul TE buffer to your labeled RG57 probe (from step I). Pipette entire sample (85 ul) into the column. Centrifuge for 1.0 min at 7,000 in the small microfuge.
    6. Add 100 ul TE buffer to the column, and microfuge for 1 min.
  1. Prehybridization
    1. Make up enough hybridization solution for your experiment (We use about 25 ml per blot).
    2. Insert membrane into a hybridization tube and add hybridization buffer.
    3. Incubate at 65° C in the hybridization oven for about 1 hour.

    IV. Hybridization

Do all of the following behind a shield

    1. Heat your probe at 95° C for 5 min (to denature it) and immediately place on ice for 5 min.
    2. Add the denatured probe mix directly to the hybridization tube.
    3. Hybridize overnight in the oven at 65° C.
    4. Pour hybridization solution in liquid radioactive waste.
    5. Add 50 ml 2.0X SSC, 0.1% SDS to the tube and wash the membrane for 15 min at 65° C. Pour off wash solution into radioactive waste.
    6. Add 50 ml 0.5X SSC, 0.1% SDS to the tube and wash for 15 min at 65° C. Pour off wash solution into radioactive waste.
    7. Repeat step 6.
    8. Carefully remove membrane from tube and wrap with saran wrap.
    9. Place membrane in a film cassette in contact with X-ray film. Expose overnight.

Solutions for Radioactive Detection

50X Denhardt’s solution

5 g Ficoll (Type 400, Pharmacia)

5 g polyvinylpyrrolidone

5 g bovine serum albumin (Fraction V; Sigma)

Add H2O to a total volume of 500 ml.

20X SSC

Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H2O. Adjust the pH to 7.0 with a few drops of a 10 N solution of NaOH. Adjust the volume to 1 liter with H2O. Dispense into aliquots. Sterilize by autoclaving.

0.5 M NaPO4 buffer pH 7.0

577 ml of 1M Na2HPO4

423 ml of 1 M NaH2PO4

Hybridization buffer (25 ml)

2.5 ml of 50 X Denhardt’s solution

6.25 ml of 20 X SSC

2.5 ml of 0.5 M NaPO4 buffer pH 7.0

0.5 ml of 0.5 M EDTA

12.63 ml H2O

0.625 ml of 20% SDS

2 X SSC, 0.1% SDS

100 ml of 20 X SSC

1 g of SDS

Add water to a total volume of 1 liter

0.5 X SSC, 0.1% SDS

25 ml of 20 X SSC

1 g of SDS

Add water to a total volume of 1 liter

infected tubers

infected tomatoes