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Standard Southern Blot Procedure

Jeffrey M. Leiden, M.D., Ph.D.

1. Digest 10 ug high molecular weight DNA in a total volume of 45 ul with 50-100 units of restriction enzyme and 1/10th volume of the appropriate 10X RE buffer 4 hours-overnight at 37°C. Add 1 ul of RNase for the last 1 hr of digest.

2. Pour a 350 ml 0.8-1% agarose gel in TAE buffer with 17.5 ul ethidium bromide (10 mg/ml) using a comb containing 20 2 mm x 1 cm wells.

3. Add 1/10 volume (5 ul) of 10X DNA loading buffer to each sample.

4. Load gel and include 15 ul of premixed size markers (lambda HindIII/uX174 HaeIII) in both of the outside lanes.

5. Run the gel at 120 V for 5-6 hours at room temperature. Recirculate buffers at 3 hr.

6. Photograph the gel with a ruler next to the size markers and cut off the upper left hand corner of the gel. Soak the gel for 1 hour with gentle shaking in 300-500 ml of denaturation buffer at room temperature.

7. Pour off denaturation solution and rinse the gel with a small amount of renaturation solution.

8. Soak the gel for 2 x 30 minutes at room temperature in 500 cc of renaturation buffer with gentle shaking.

9. Put on disposable gloves and set up a standard blotting chamber using 10X SSC as the transfer buffer

10. Cut a piece of .45 micron nitrocellulose (Schleisher and Schuell) to exactly the size of the gel. Keep hands off of nitrocellulose. Drop the nitrocellulose onto the surface of a glass dish containing doubly distilled water and wet completely. Then submerge the nitrocellulose in the water.

11. Flip the gel over (so bottom of gel is facing up) and place onto the Whatman paper on the bottom of the blotting apparatus. Note: place 4 sheets of Whatman on top of the blotting stone.

12. Using a spatula surround the gel with pieces of parafilm which underlap the gel by 1 mm on each side.

13. Transfer the nitrocellulose to a dish containing 10X SSC for 30-60 seconds.

14. Place the nitrocellulose on top of the gel and roll out any bubbles with a piece of disposable 5 ml pipet. Cut 2 pieces of Whatman 3 MM paper to exactly the size of the gel and quickly soak in 10X SSC before placing on top of the gel. Roll out bubbles with the 5 ml pipet.

15. Cut a stack of paper towels to the size of the gel and place on top of the Whatman strips.

16. Place the top on the blotting apparatus and weight down with a 0.5 kg weight or a 500 cc bottle filled with water.

17. Blot overnight.

18. Put on gloves. Remove and discard paper towels and Whatman filters. Carefully remove the nitrocellose sheet with the gel in position and flip over onto a clean glass plate. Mark the wells with a #2 pencil and remove and discard the gel. Cut upper left hand corner of nitrocellulose.

19. Soak the nitrocellulose briefly (1-2 minutes) in 2X SSC.

20. Place nitrocellulose face up on a piece of dry Whatman paper and air dry 15-20 minutes.

21. Place the nitrocellulose between 2 pieces of precut Whatman paper and tape shut into a folder. Bake for 2 hours at 80°C in a vacuum oven.

SOLUTIONS:

Denaturation Solution: 1.5 M NaCl Store R.T.
0.5 M NaOH

Renaturation Solution: 1.0 M Tris.Cl (pH 8.0) Store R.T.
1.5 M NaCl

20X SSC: 175.3 g NaCl
88.2 g NaCitrate (trisodium salt), pH to 7
H2O to 1 L

Ethidium Bromide: 10 mg/ml in water. Stir 6-12 hours. Store 4°C in brown bottle.

50X TAE: Trisma base. 242 g/L.
Glacial Acetic Acid: 57.1 ml/L.
O.5 M EDTA (pH 8.0): 100 ml. Store R.T.

10X Loading Buffer: 0.42 % Bromophenal blue Store at 4°C
0.42 % xylene cyanol
50% glycerol in water

Size Markers: 50 ug/ml HindIII cut mlDNA in T.E Store -20°C

50 ug/ml HaeIII cut rX174 DNA in T.E.

1/10 volume 10X loading buffer

Solutions:

1. tRNA: yeast tRNA (Sigma) in H2O at 10 mg/ml. Store 4°C.

2. STE: 10 mM Tris (pH 7.5) Store R.T.
1 mM EDTA
100 mM NaCl
f

C. PreHybridization Reaction:

1. Place nitrocellulose blot in a seal-a-meal bag and seal 0.5 cm from each edge on 3 sides leaving the top open.

2. Prepare 25 ml of hybrization cocktail as follows:
Fluka Formamide 12.5 ml
20X SSC 6 ml
1 M Tris pH 7.5 0.2 ml
50X Denhardt's Solution 2.5 ml
10% SDS 0.5 ml
50% Dextran Sulfate (Oncor) 2.5 ml
Water to 25 ml

3. Boil 0.25 ml of salmon sperm DNA (10 mg/ml in TE) for 7 minutes in an eppendorf tube.

4. Quick freeze both the salmon sperm DNA on dry ice.

5. Thaw the the salmon sperm DNA at 68°C quickly.

6. Add the salmon sperm DNA to the premixed and warmed hybridization buffer and mix well.

7. Add the hybridization cocktail to the Southern blot in the seal-a-meal bag. Avoid bubbles in the hybridization bag by rolling them out with a 10 ml pipette on top of a glass plate. Make sure that the blot is uniformly wet.

8. Prehyb at 42oC for 2 hrs.

C. [32P]-Labelling of Probe:

1. Add 25 ng of DNA to a total volume of 10 ul of H2O in a microfuge tube.

2. Boil x 10' and place on ice. Spin down 10 sec in a microfuge

3. Immediately add:
3 ul of dATP, dGTP, dTTp mixture - Thaw rapidly and
2 ul solution 6 (reaction mixture) - keep on ice
5 ul [a32P] dCTP (3000 Ci/mM)-must have reference date within 1 wk of labelling
1 ul Klenow enzyme (5 U/ml)

4. Incubate 37°C x 45'

5. Add 2 ul 500 mM EDTA and place at 65°C for 10' to stop reaction.

6. Prepare spin column: 1) Place G-50 spin column (Boehringer) and collection tube in 15 ml polystyrene tube

2) Spin 3000 RPM x 2'

3) Discard collection tube and replace it with another in polystyrene tube

7. Add 2 ul yeast tRNA (10 mg/ml in H2O) and 50 ul STE to hexapriming mixture and mix.

8. Load hexapriming mix onto spin column

9. Spin 3000 RPM x 4-5'

10. Discard column and place effluent in eppendorf tube.

11. Count 1 ul of effluent to determine the specific activity of the probe. The specific activity should be at least 1 x 109 DPM/ug DNA

12. Boil probe x 10' and place on dry ice prior to use.

D. Hybridization

1. Add the probe to the edge of the prehybridization solution without squirting directly onto the blot by pipetting and mix probe into solution so that it is uniformly spread over the blot.

2. Place the seal-a-meal bag on a glass plate and gently roll out all bubbles with a 5 ml disposable pipet. Seal the final side of the seal-a-meal bag 0.5 cm from the top of the blot.

3. Wash the seal-a-meal bag well with running water and place in a 42o C water bath overnight.

4. In the morning remove the blot from the bag directly into 500 cc of 2X SSC/0.1% SDS. Wash with gentle shaking at room temperature for 10 minutes. Repeat the 2X SSC/0.1% SDS wash for an additional 10 minutes at room temperature with gentle shaking.

5. Rinse the blot with a small volume of 0.1X SSC/0.1% SDS at room temperature for 10 seconds and discard.

6. Add 500 cc of 0.1X SSC/0.1% SDS to the blot in a tupperware container. Seal the top and place at 65°C for 30 minutes. Repeat this 65°C wash for an additional 30 minutes.

7. Remove the blot onto clean sheet of Whatman paper and listen to it with a Geiger counter. It should not be hot.

8. Let the blot air dry until surface liquid is gone, but the blot is still damp.

9. Wrap the blot in saran wrap and tape to a clean sheet of Whatman paper which has been marked asymmetrically with dots of radioactive ink.

10. Autoradiogram using an intensifying screen at -70°C for 4-24 hours.

E. Stripping Southern Blot on Nitrocellulose:

Treat blot with:

  1. 0.5 N NaOH x 10 min. at R.T. (250 ml)
  2. 0.5 N NaOH x 10 min. at R.T.
  3. 1 M Tris pH 7.6 x 10 min. at R.T. x 2

Dry blot slightly on Whatman, immediately place into hybridization cocktail.

 

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