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S U P P O R T
 

Protocol for Southern Blotting of Genomic DNA
(with DNA Marker for Genomic DNA Analysis)

I. Solutions required (see below)
II. Electrophoresis
III. Southern blotting
IV. Marker probe generation
V. Hybridization
VI. Autoradiography

I. Solutions required
  1. Tris-acetate buffer: Fermentas 50X TAE diluted to 1X concentration.
  2. Denaturation solution: 1.5 M NaCl, 0.5 M NaOH.
  3. Neutralization solution: 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2), 1 mM EDTA.
  4. 20X SSC solution (blotting buffer): 3 M NaCl, 0.3 M sodium citrate, 1 mM EDTA.
  5. 100X Denhardt's solution: 2% (w/v) BSA (bovine serum albumin), 2% (w/v) Ficoll, 2% (w/v) PVP (polyvinylpyrrolidone).
  6. Hybridization solution: 5X SSC, 5X Denhardt's solution, 40% formamide, 0.5% SDS.
  7. 1 mg/ml sonicated herring sperm DNA solution.
  8. 10% (w/v) SDS solution.

II. Electrophoresis

Dilute 1 µl of DNA Marker for Genomic DNA Analysis with 39 µl of nuclease-free water and mix. To 10 µl of this mixture (50 ng of marker per gel lane or approx. 6 ng of marker per 1 mm of gel lane width) add 1.5 µl of 10X DNA Loading Dye and 3.5 µl of nuclease-free water and run in a 20 cm 0.7% agarose gel in 1X TAE buffer for 18 hours at 3 V/cm (until bromophenol blue marker reaches the bottom of the gel).

Note

The cohesive ends of the 12 nt cos sites of bacteriophage lambda that appear in two separate fragments may anneal and form an additional band. Prior to loading on gel, these fragments can be separated by heating at 65°C for 5 min and then chilled on ice for 3 min.

III. Southern blotting
  1. Before blotting, rinse the gel in deionized water and shake in the denaturation solution for 30 min at room temperature. Rinse the gel in deionized water and shake in the neutralization solution for 15 min at room temperature. Repeat procedure.
  2. Fill the glass dish with 20X SSC blotting buffer. Make a platform and cover it with a sheet of Whatman™ 3 MM filter paper, saturated with blotting buffer (as shown in picture).
  3. Place the gel upside down on the wick and avoid trapping air bubbles beneath it.
  4. Cut a sheet of SensiBlot™ Plus Nylon Membrane to match the size of the gel and place it on the top of the gel. Avoid trapping air bubbles beneath the membrane.
  5. Place 2-3 sheets of Whatman™ 3 MM filter paper cut to size and wetted with blotting buffer on the top of SensiBlot™ Plus Nylon Membrane.
  6. Place a stack of absorbent paper towels on top of the 3 MM paper, place a glass plate on the top of the paper towels and put a 0.5 kg weight on the top.
  7. Perform upward capillary transfer of DNA at room temperature for 18 h (as shown in picture).
  8. Wash the membrane in 2X SSC solution to remove any residual agarose, dry at room temperature and fix for 2 min under UV-light.

IV. Marker probe generation

50 ng of radioactively labeled marker is sufficient for 3-5 hybridization reactions. Use the same protocol for labeling of the genomic DNA hybridization probe (test probe).

  1. Prepare the following reaction mixture:
    DNA template 5 µl (50 ng)
    Hexanucleotide in 5X reaction buffer 5 µl
    Water, nuclease-free to 20 µl
  2. Vortex the tube and spin down for 3-5 s. 
  3. Incubate the tube in a boiling water bath for 5-10 min and chill on ice. Spin down quickly.
  4. Based on your choise of labeled triphosphate (dATP or dCTP) use Mix A or Mix C, respectively.
  5. Add the following components to the same tube:
    Mix A or Mix C (supplied with the kit) 1.5 µl
    [alfa-32P]-dATP or [alfa-32P]-dCTP (0.925 MBq = 25 µCi) 3 µl
    Klenow Fragment, exo- (supplied with the kit) 0.5 µl (2.5 u)
  6. Shake the tube and spin down in a microcentrifuge for 3-5 s. Incubate for 10 min at 37°C.
  7. Add 2 µl of the dNTP Mix and incubate for 5 min at 37°C.
  8. Stop the reaction by addition of 0.5 µl of 0.5 M EDTA, pH 8.0.

V. Hybridization
  1. Prepare 25 ml of the hybridization solution.
  2. Denature 0.5 ml of sonicated herring sperm DNA solution by heating to 100°C for 5 min. Chill on ice and add to the hybridization solution.
  3. Put the membrane into the hybridization bag and carry out the prehybridization with 12 ml of the hybridization solution for 2 hours at 42°C.
  4. Use a mixture of both genomic DNA marker labeled probe and genomic DNA hybridization labeled probe (test probe).
  5. Denature labeled probes by heating to 100°C for 5 min and chill immediately on ice.
  6. Add 10-15 ml of the hybridization solution and 1/5 of the labeled probe to the hybridization bag.
  7. Incubate for at least 12 hours at 42°C.
  8. Carry out the following washes of the membrane:
    Twice for 10 min in 2X SSC + 0.1% SDS at room temperature
    Twice for 15 min in 1X SSC + 0.1% SDS at 65°C
    Twice for 10 min in 0.1X SSC + 0.1% SDS at 65°C
  9. Dry the membrane using sheets of Whatman™ 3 MM paper.
VI. Autoradiography

Wrap the dried membrane with Saran Wrap™ and expose to a phosphoimager or a film with an intensifying screen.

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Updated vasario 01, 2008 09:30