Experiment 3:
Southern Blotting
Theory:
A Southern blot is a method routinely used in Molecular Biology for detection of a
specific DNA sequence in DNA samples. Southern blotting combines transfer of
electrophoresis - separated DNA fragments to a solid support (either nitrocellulose filters
or nylon membranes), and subsequent fragment detection by probe hybridization. After
pre-hybridization to reduce nonspecific, hybridization with the probe, the filter or
membrane is hybridized to the desired radiolabeled chemical labeled nucleic acid probe.
The filter or membrane is washed to remove unbound and weakly binding prob and then
autoradiograph.
The method is named after its inventor, the British Biologist Edwin Southern. Other
blotting i.e., Western blot and Northern blot, that employ similar principles but using
RNA or protein, have later been named in reference to Edwin Southern's name.
Materials Required:
Electrphoretic chamber, power supply, cross linker apparatus, trays, UV lamp, polaroid
camera, x-ray machine, x-ray cassette, x-ray film, agarose, denaturation solution (1.5 M
NaCl and 0.5 NaOH), Neutralization so;ution (1.5 M NaCl and 1 M Tris base), 10x SCC
buffer (3 M NaCl and 0.3 M Na-citrate), prehybridization solution (12.5 ml of 1 M
KPO4, pH 7.4, 125 ml of 20x SSC, 25 ml of 100x denhardt’s or nonfat powdered milk, 5
ml of 5 mg/ml salmon sperm DNA 250 ml 100% formamide, 1% SDS in 82.5 ml H 2O).
Labelled prob for hybridization (previous protocol), Palstic trays, nitrocellulose filters, 46
x 57 cm whatman 3MM paper, sealable bags, sealer.
Procedure:
1. After separation of DNA fragments on agarose photograph it with a Polaroid camera.
If ruler is placed alongside the gel when it is photographed, a graph of the log
molecular weight versus mobility of the unlabeled marker fragments can be drawn.
2. Transfer the gel to a tray and trim the unused area of the gel, cut one corner to mark
the orientation, that is the start point.
3. Denature the gel at room temperature for 30 minutes with shaking in denaturation
solution.
4. Neutralize the gel at room temperature for 30 minutes with shaking in neutralization
solution.
5. In the mean time, prepare a wick by cutting one piece of whatman 3 MM ~2 cm wider
than the width of the gel and 30 to 40 cm long according to the size of the gel box.
Pour several hundred 10x SSC in the gel box. Put the glass plate the box and place
wick on it with both ends of wick hanging over plate into the 10x SSC. Remove air
bubbles trapped between the wick and glass plate by rolling 10 ml pipette back and
forth over wick.
6. Lift the gel out of neutralization solution, allow most of the liquid to drip off the gel
and lay gel on the top of the Whatman 3MM wick. Remove air bubbles trapped
between gel and wick by rolling a pipette over gel as above. Remove the
nitocellulose (NC) filter from its tray of 10x SSC and lay it on top of the gel, making
sure that the NC filter does not overhang the gel.
7. Wet one piece of the stack of cut Whatman 3MM paper in 10x SSC and place it on
the top of the NC, make sure no air bubble trapped in. Place the stack of cut papers on
top of the first 3MM paper then put 2-3 cm thick stack paper towels on top of 3 MM
papers. To keep the entire pyramid pressed together, place a glass plate on top and a
small bottle on the glass plate.
8. Cover up the ends of the gel box with plastic wrap to minimize evaporation during
the transfer and allow the transfer to proceed for 12 hours.
9. After the transfer is complete, take a part the pyramid so that the NC filter is still
lying on the gel. With a blue ball point, mark on the filter the location of the slots.
Using blunt forceps, remove the NC paper and cross linked for 1 minute in a cross
linker apparatus or place the dried filter between two pieces of 3MM paper and bake
the filtr for 30 minutes to two hours at 80o C in a vacuum oven.
10. Put the NC paper in a sealable bag and pre-hybridize for 2-3 hours at 50o C. Cut the
bag from one corner, add labeled probe ( at least 0.5x106 cpm/ml) and seal the bag
again.
11. Hybridize for 3-5 hours (overnight in case of oligos) at 50o C.
12. Cut the bag, remove NC and put in a tray and wash with shaking as follows: 2x SSC
for 10 minutes (2x), 0.5x SSC for 10 minute (2x) at 50o C. Check the back ground
with giger counter.
13. Rap in plastic wrap and expose the filter to x-ray film at 70o C for 3-4 hours
depending upon the signal.
http://www.dnatube.com/video/1512/Southern-blot