Southern Blot

DNA transfer from an agarose gel to a nylon membrane.
  1. Take a photo of the gel.
    Cut the extra gel- upper part of the gel, just above the wells and all unused lines. Measure gel’s dimensions in order to prepare the nylon membrane and 3mm paper sheets.
    Place two fluorescent rulers on both lateral sides of the gel and level the beginning of the ruler’s cm scale with the well line. Make sure that on the photo you can see well the ruler’s scale, the marker and all the visible DNA bands of your samples. If necessary, make several photos with different exposure time in order to get the whole information and then combine them in your notes.
  2. Depurination of the DNA.
    Incubate the gel in 0.125N HCl for 15min@RT with moderate shaking.
  3. Denaturation.
    Rinse the gel 3X with dH2O and then add the denaturation buffer.
    Incubate 30min@RT/moderate shaking.
  4. Neutralization.
    Change the denaturation buffer with the neutralization buffer and incubate the gel for further 30min with moderate shaking.
    In the meantime, cut 3 sheets of 3MM paper and the nylon membrane in the size of the gel.
  5. Transfer assembly.
    Assemble first the sponges and wet them with 20X SSC.
    Wet 2 sheets of 3MM in 20X SSC and put them onto the sponges. Make sure that there are no air bubbles between the sheets and sponges.
    Put the gel face up on the 3MM paper and cut the upper left corner (the beginning of the gel).
    Pre-wet the membrane first in dH2O and then in 20XSSC. Place the membrane on the gel. Roll a clean pipette over the gel to get out all the air bubbles. Wet another sheet of 3MM paper and put it on the membrane. Get out all the air bubbles with a clean pipette.  
    Surround the gel and the membrane with saran wrap to avoid short cut buffer transfer.
    Put a tall layer of paper on top of the 3MM paper and finally, press the paper with a ~1kg weight.
    Leave the transfer ON@RT.
Fixing of DNA to a nylon membrane.
Discard wet paper towels and 3MM paper.
Mark the wells of the gel on the membrane with a water and alcohol proof marker.
Cut the corner of the membrane where the first DNA sample was loaded (the beginning of the gel).
Leave the membrane on a clean and dry sheet of 3MM paper to air dry, and then bake it @80oC for at least 1 hour.
After the baking, wash the membrane for 15 minutes at RT in 2XSSC.
 
Prehybridization.
Thaw an aliquot of ssDNA (salmon sperm DNA, white box with yellow tape in Laura’s freezer).
Put your membrane in a hybridization tube. Pour 10-12ml of the hybridization buffer in a big tube or 8-10ml in a medium one.  Rotate the membrane at 65oC until they warm up.
Denature ssDN 5’/95oC. Place the tube on ice and then spin it briefly at 4oC.
Add denatured ssDNA to the hybridization buffer.
Prehybridize the membrane(s) for at least 1 hour.
 
Probe labeling with 32PdCTP.
Use Rediprime kit for labeling.
Thaw the tube with 32PdCTP under the radioactive hood.
Denature 30-50ng of an appropriate probe in Vtotal= 45μl  (5’/95oC). Place the sample(s) on ice and then spin them down briefly. Add this denatured DNA to a desiccated labeling mixture (Rediprime), and then finally add 5μl of dCTP.
Mix the sample well by pipeting it up and down until it turns uniformly light blue.
Leave it at 37oC at least for 30min.
Prepare a G-50 Sephadex column for each sample.
Purify the sample(s) on a Sephadex G-50 column.
Collect 50μl of the radioactively labeled and purified probe. Denature the probe 5’/95oC, place it on ice to snap cool and then spin it briefly at 4oC. Add the denatured probe to the prehybridization mixture. Hybridize ON.