Southern Blot

From CCGB

HardisonProtocols

Southern

    -- by Hao Wang

Transfer

Materials:

1 nylon membrane cut to size of gel (usually 15×20cm for large gel).

2 pieces of whatman paper cut to size of gel

10 blot pads

1. Soak nylon membrane in dH2O for at least 10min.

2. Soak one blot pad in 0.4N NaOH for 15min.

3. Pour out dirty NaOH

4. Add fresh NaOH and nylon membrane to blot pad

5. Soak for 15min

6. Put both pieces of filter paper in NaOH until wet.

7. Use plastic container with lid and sponge.

8. Squeeze out sponge to remove any old NaOH.

9. Saturate sponge with fresh 0.4N NaOH until excess can be seen.

10. Put approximately 200ml 0.25M HCl into glass baking dish.

11. Carefully put gel in HCl.

12. Gently shake for 30min.

13. Carefully pour off HCl.

14. Rinse gel in dish with dH2O 3 times.

15. Lay soaked blot pad on top of saturated sponge.

16. Carefully place gel well-side down on blot pad. Roll with glass rod to remove air bubbles.

17. Place soaked nylon membrane on top of gel. Roll with glass rod to remove air bubbles.

18. Put whatman paper on top of nylon membrane one sheet at a time. Roll with glass rod.

19. Place 9 dry blot pads on top of filter paper in line with gel.

20. Put 2 glass plates on top of 9 blot pads.

21. Put lid on container.

22. Allow transfer to take place 4-6 hrs to overnight.

23. Mark wells with pencil. Dry the nylon membrane @80°C for 1 hr. Crosslink.

Prehybridization and Hybridization

Prehybridization

1. Preheat LAB-LINE Incubator/shaker to 75°C. It will take about 1 hour to reach 75°C.

2. In a large rubber maid container place up to 8 membranes, prewetting those that are dry. Cover membranes with 100-300 ml prehybridization solution.

 Add in order:	14.5 ml ddH2O

50ml 1M NaPO4 pH7.2 35ml 20% SDS 0.3ml 0.5M EDTA pH8.0 1g BSA Stir with gentle heat

3. When dissolved, add 1 ml salmon sperm (10mg.ml) which has been boiled for 10 min for 100 ml. Stir well. Pour solution over membranes, making sure solution gets between each membrane. Cover box tightly and let the membranes swirl gently overnight.

Hybridization

1. Prepare probe and label it with 32P. While the probe is boiling for 5 min after going through the column, prepare the membranes.

2. Wrap membrane around a 25ml pipet and place the wrapped end into a clean hybridization tube. Unwrap the membrane carefully tightly against the sides of the tube.

3. Start with the ends of the membranes pressed flush against the glass and unroll them slowly, holding the pipet firmly against the glass as you unroll and turn the tube at the same time.

4. Draw 6-12 hybridization solution into the tube.

5. Take the probe out of the boiling water bath, cool on ice for 5 min.

6. Draw probe up and dispense it into the tube being very careful. Put lid on tightly and swirl solution.

7. Place tube in chamber with balancing tube. Close glass door and let tubes rotate. Check to see that fluid is moving from bottom to top of tube like a wave. Close door and let the membrane hybridize overnight.

Hybridization Solution (50ml)

Stir	7.3ml ddH2O 
       25ml 1M NaPO4 pH7.2
       17.5ml 20% SDS
       150μl EDTA pH8.0

Washing membrane after labeling

 Wash Solution I (1 liter)
     40ml 1M NaPO4
     2ml EDTA pH8
     50g SDS
 Stir with heat until dissolved.

 Wash solution II (1 liter)
     40ml 1M NaPO4
     2ml EDTA pH8
     10g SDS
 Stir with heat until dissolved.

  1M NaPO4 (1 liter)  
  Na2HPO4		97.2g
  NaH2PO4		43.6g
  Stir with heat till dissolved. PH to 7.2

1. Pour the hybridization solution out of the tube into the liquid radioactive waste container. Catch the drop with a kimwipe; discard the wipe in the solid waste. Rinse out tube with 25-50ml Wash I.

2. First Wash: Heat Wash I to 65°C. Gently rock the tubes till the membranes are eased to the mouth of the tube. Dump the rinse into the waste container. Grasp the edge of the curled membrane with forceps or gloves and pull them out slowly into ta Rubbermaid container. A maximum of 8 membranes can be washed at one time in a container. Use a minimum of 100 ml Wash I for 1 membrane up to 300-350ml for 8. unfold membranes. Pour Wash over membranes carefully. Make sure the membranes are free floating. Shake gently at 65°C for 15-30 minutes.

3. Consecutive washes: Heat Wash II at 65°C. Usually takes three washes of 10-15 min each. When the wash comes off cold, pour it down the drain, blot the membranes dry and wrap them individually in plastic wrap.

4. Expose to PhosphoImager at room temperature overnight.