| Contributor: |
The Laboratory of Jasper Rine at the University of California, Berkeley
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| Southern blotting provides a means for determining the existence of particular sequences or genes within genomic DNA. This protocol has been optimized for yeast DNA. |
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A. Agarose Gel Electrophoresis
1. Electrophoresis DNA on an Agarose gel with Ethidium Bromide and DNA size markers (CAUTION! see Hint #1). Photograph the gel next to a ruler (see protocol on Agarose Gel Electrophoresis of DNA; also see Hint #2)
2. Depurinate gel in 0.2 M HCl for 10 min (see Hint #3).
3. Denature for 30 min in several volumes of Chloride/Hydroxide Solution.
4. Neutralize denaturing solution for 45 min in several volumes of Tris/Chloride Solution.
5. Transfer gel to 10X SSC for 8 to 24 hours (see Hint #4).
B. Transfer Apparatus
1. Fill the bottom of the transfer apparatus with 10X SSC.
2. Lay a glass plate across the width of the dish and drape a pre-wetted 3MM Filter Paper across the plate (pre-wet in 10X SSC). Make sure the edges of the filter paper are submerged in buffer.
3. Smooth out all bubbles between the paper and the glass plate with a glass rod.
4. As an alignment guide, remove a small section of the lower left hand corner of the neutralized gel.
5. Invert the gel and place it on the 3MM Filter Paper.
6. Lay Saran Wrap on top and cut out the Saran Wrap that is covering the gel (see Hint #5).
7. Cut Hybond N™ membrane such that it is 1 mm larger than the gel in both dimensions.
8. As an alignment guide, remove a small section of the lower left hand corner of the Hybond N™ membrane.
9. Wet membrane with ddH2O and soak for at least 5 min in 2X SSC.
10. Lay the membrane over the gel making sure there are no bubbles between the filter paper and the gel.
11. Wet 2 pieces of 3MM Filter Paper (cut to exactly the same size as the gel) in 2X SSC and place them
on top of the wet nitrocellulose membrane.
12. Smooth out air bubbles and then place 5 to 10 paper towels on top of the 3MM Filter Paper.
13. Put a glass plate on top of the stack and weigh it down with a 500 g weight.
14. Transfer overnight at room temperature.
15. Remove the paper towels and the 3MM Filter Paper.
16. Lay the gel down, gel side up, on a dry sheet of 3MM Filter Paper.
17. Mark the positions of the gel slots on the Hybond N™ membrane with a soft-lead pencil
18. Peel the gel from the membrane and discard the gel.
19. Soak the membrane in 6X SSC for 5 min at room temperature to remove excess agarose.
C. Cross Linking by UV
1. Immediately transfer the membrane to a Stratalinker and expose to UV light (see Hint #6).
D. Cross-Linking by Baking
1. Allow excess SSC to drain from the membrane by laying on a paper towel for at least 30 min.
2. Sandwich the membrane between two pieces of 3MM filter paper.
3. Fix DNA to the membrane by baking for 90 min at 80°C in a vacuum oven.
E. Hybridization
1. Wet membrane with 6X SSC.
2. Place membrane in Hybridization Buffer and allow to soak for approximately 5 min.
3. Boil 100 μl of the radiolabeled probe for 5 min (CAUTION see Hint #1).
4. Cool boiled probe on ice for 5 min.
5. Dilute to 1 ml in Hybridization Buffer and mix well by inversion.
6. Add diluted probe mixture to hybridization bath containing membrane.
7. Hybridize overnight at room temperature.
8. Remove the Hybridization Buffer and wash the filter for 30 min at room temperature in 2X SSC/SDS.
9. Remove the SSC/SDS solution and wash the filter in 0.2X SSC/SDS for 20 min at 65°C (see Hint #7).
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| 0.2X SSC/SDS |
| 0.1% (w/v) SDS
0.2X SSC
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| 2X SSC/SDS |
| 0.1% (w/v) SDS
2X SSC
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| Hybridization Buffer |
| 0.5% (w/v) SDS
1X Denhardt Solution
6X SSC
0.1 mg/ml of sonicated Salmon Sperm DNA
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| Denhart Solution |
| 10 g Polyvinyl pyrrolidone
10 g Ficoll 400
10 g Bovine Serum Albumin (fraction V)
Store at -20°C in aliquots
Bring the final volume to 500 ml using ddH2O
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| SSC (10X) |
| pH 7.2
3 M NaCl
0.3 M Sodium Citrate
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| Tris/Sodium Chloride |
| 1 M Tris, pH 7.4
1.5 M NaCl
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| Chloride/Hydroxide Solution |
| 1.5 M NaCl
0.5 M NaOH
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| 0.2 M HCl |
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SDS
Agarose
Tris
Bovine Serum Albumin (BSA), Fraction V
Ficoll 400
Oligonucleotide
Hydrochloric Acid
Ethidium Bromide
Sodium Citrate
DNA, Salmon Sperm
Sodium Chloride
Polyvinyl Pyrolidone
Sodium Hydroxide
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. To detect 1.0 Kb to 3.6 Kb shift use a 0.85% Agarose gel.
3. Depurination is usually done if fragments of interest are greater than 15 Kb. Depurination can be done for smaller fragments but bands may be fuzzy and overpurination of small fragments can lead to poor transfers.
4. Sixteen hours is a good transfer time for fragments between 1.0 Kb to 3.6 Kb; however, you may want to determine the transfer time empirically.
5. The hole allows access to the gel for the nitrocellulose paper.
6. While cross linking the DNA to the membrane, make sure that the membrane doesn't dry out.
7. Lower salt concentration will increase the hybridization stringency.
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