Southern Blot analysis to characterize transformants.
The Southern blot analysis is useful for identifying different lines, determine insert copy number, and for detecting any gross DNA rearrangements that might have occurred during transposition.  If you are analyzing a ß-galactosidase containing insert, digestion with EcoRV might be appropriate as this cuts about 1kb upstream and downstream from where the ß-galactosidase coding sequence begins.

Restriction digestion, gel analysis and blotting.  This is a standard Southern Blotting procedure. GeneScreen Plus (NEN Life Sciences) will be used as a blotting membrane.

  1. Digest at least 1 ug of Drosophila genomic with appropriate restriction enzymes. Digest 1 to 5 ug of DNA in a volume of 20 ul with 10 units of enzyme. Allow the digestions to go for 4 to 12 hours.
  2. Check the quality of the digestion by analyzing 100 ng of each sample on a minigel. Include an undigested control sample for comparison. The digested samples should appear as a smear and bands from repetitive sequences might be evident.
  3. Run the remain DNA on a 1% agarose gel. Include a lane with lambda markers for when the gel is stained with ethidium bromide and photographed.
  4. Stain the gel for 20 minutes in a solution of 1ug/ml ethidium bromide. Destain in water for 10 minutes and photograph.
  5. Soak the gel for 15 minutes in 0.12 M HCl - the bromophenol blue should turn yellow.
  6. Soak the gel in 0.4 M NaOH for 30 minutes.
  7. Cut three pieces of blotting paper that will extend about 1 inch beyond the gel on all four sides. Soak each of these with 0.4 M NaOH and stack them in a glass tray. (See the diagram later in this section for an illustration of how the blot will be assembled.)
  8. Cut the gel through the middle of the wells and discard the top portion. By cutting through the middle of the wells, indentations will remain at the top of the gel when the gel is flattened by the blotting procedure, and these can be used to mark on the filter where the wells were located on the gel. Flip the gel upside down and place it in the middle of the stack of blotting paper. Be certain to push out any air bubbles. The gel is flipped because the DNA is usually closer to the bottom side of the gel.
  9. Frame the gel with strips of plastic. The plastic should extend under the edges of the gel by 1 to 2 millimeters. Gently force air bubbles out from under the gel. The plastic prevents the buffer from flowing around the gel.
  10. Cut a piece of GeneScreen Plus to match the size of the gel. Wet the surface of the gel with several drops of 0.4 M NaOH. Slowly lay the gene screen onto the surface of the gel; the filter should wet readily. Avoid trapping air bubbles under the filter. Also avoid shifting the filter once it has contacted the gel as some DNA may have already begun to transfer to the filter.
  11. Cut 2 pieces of blotting paper to match the GeneScreen and lay one dry sheet at a time on top of the filter. Allow the first paper to wet, and smooth it out before adding the second. The second piece does not have to wet completely before assembling the stack of paper towels.
  12. Pile a 1 inch layer of paper towels on top of the top piece of blotting paper. The towels should be cut to match the size of the filter.
  13. Place a flat piece of plexiglass or glass on top of the paper towels. Weigh the stack down with a bottle containing 200 to 500 ml of any solution.
  14. Allow the DNA to transfer for at least 6 hours. Overnight transfers are typical.
  15. After transfer, remove the filter and soak it for 15 minutes in 200 ml of 0.2 M Tris-Cl pH 7.5, 2X SSC.
  16. Blot the filter dry with paper towels and then allow it to air dry for at least 1 hour. The filter can be stored in this dry state. Air-drying is sufficient to permanently fix the DNA to the filter. There is no need for baking the GeneScreen Plus filter to attach the DNA.

Preparation of radiolabeled DNA for probing the Southern blot.  Several different manufacturer's supply kits for radiolabeling probes. Kits based on random priming generate radioactive probes with specific activities of approximately 109 cpm/ul. A gel-purified DNA fragment will be provided for the random priming reactions. Perform the radiolabeling reactions according to the specifications of the manufacturer.

Since most random priming reactions are essentially the same, the following procedure can be used to remove unincorporated radioactive nucleotide.

  1. Stop the reaction by adding 0.5 M EDTA to a final concentration of approximately 20 mM.
  2. Add 400 ul of TE, 1 ul of 5 mg/ml salmon DNA and 20 ul of 100 mM spermine. Mix well.
  3. Incubate on ice for 15 minutes, centrifuge for 15 minutes and discard supernatant.
  4. Dissolve pellet at 37oC in 50 ul of 0.5 M NaCl/TE. Allow 10 minutes to dissolve.
  5. Add 200 ul of TE and count a small amount to determine the efficiency of labelling the DNA. Store the probe in the freezer.
I.F. Probing the Southern Blot with radiolabeled probe.
The filter is first soaked for a short period in hybridization mix minus probe. Then probe is added directly to the hybridization solution. There is no special prehybridization mix.
  1. Roll the blot into a cylinder so the DNA side is towards the center of the cylinder and place the blot in a 50 ml tube.
  2. Prepare approximately 10 ml of hybridization mix (minus probe) by boiling 200 ul of 5 mg/ml sonicated salmon DNA for 5 minutes and thoroughly mix this with 10 ml of 6X SSC, 50% Formamide, 1% SDS, 10% Dextran Sulfate. (30 ml of the solution lacking the salmon DNA can be made by putting 9 ml of 20XSSC, 15 ml of Formamide, 3 ml of 10% SDS, 3 g of Sodium Dextran Sulfate into a 50 ml tube and rotating the mixture in the hybridization oven at 42oC for approximately an hour. Store the mixture at -20oC and warm to 42oC before each use.)
  3. Add the hybridization mix to the tube containing the blot, cap the tube, and rotate the tube in the hybridization oven at 42oC for at least 15 minutes.
  4. Boil 5 to 10 million cpm’s of probe in a volume of 200 ul of TE for 5 minutes and then place the probe on ice.
  5. Remove the tube containing the blot from the hybridization oven and add the probe to the hybridization solution. Recap the tube, mix and return the tube to the hybridization oven. Rotate the tube overnight at 42oC.
  6. The next day, pour the radioactive hybridization mix into a fresh tube for reuse if needed. Store the hybridization mix in the freezer. (To reuse the probe, boil the mixture for 15 minutes before adding it to a blot.)
  7. Prepare a total of 200 ml of 2X SSC, 0.5% SDS for washing the blot. Add approximately 30 ml of this solution to the tube containing the blot, recap and rotate the tube at 25oC for 15 minutes. Discard the wash in the radioactive waste and repeat this procedure two more times.
  8. Remove the blot from the 50 ml tube and wash the blot for 15 minutes at room temperature in a tray with the remaining 2X SSC, 0.5% SDS (~100 ml). Discard the radioactive tube.
  9. Prepare 200 ml of 0.1X SSC, 0.1% SDS and warm this to 55oC.
  10. Roll the blot into a cylinder with the DNA side towards the center and put it into a new 50 ml tube. Add 30 ml of 0.1X SSC, 0.1% SDS to the blot and rotate the tube in the hybridization oven for 15 minutes at 55oC. Discard the wash and repeat this procedure until the wash solution is used up.
  11. Remove the blot from the tube and place it flat on a piece of plastic wrap. Blot away excess liquid but do not allow the filter to dry completely as it becomes impossible to strip off the radioactive probe should this be desired at a later time. Cover the filter with a second piece of plastic wrap.
  12. Expose the filter to X-ray film in the presence of an intensifying screen.