| Contributor: |
The Laboratory of Jasper Rine at the University of California, Berkeley
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A. Agarose Gel Electrophoresis of Restriction Digested DNA
1. Restriction digest the DNA and run the sample in an Agarose Gel (see protocols on Restriction Enzyme Digest of DNA and Agarose Gel Electrophoresis for DNA).
2. Take a picture of the gel with a ruler.
3. Depurinate the gel in 0.2 M HCl for 10 min.
4. Denature the gel for 30 min in Chloride/Hydroxide Solution.
5. Neutralize for 45 min in Tris/Sodium Chloride Buffer.
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 onto the pre-wetted 3MM Filter Paper.
6. Lay Saran Wrap on top of the gel and remove the section of the Saran Wrap that covers the gel (see Hint #1).
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 the membrane with ddH2O and soak it 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 the same size as the gel) in 2X SSC and place them
on top of the wet Hybond N membrane.
12. Smooth out air bubbles and then place 5 to 10 paper towels on top of the 3MM Filter Papers.
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 membrane with a soft-lead pencil.
18. Peel the gel from the membrane and discard.
19. Soak the membrane in 2X SSC for 5 min at room temperature to remove excess agarose.
20 Expose the hybridized filter to X-Ray film for an appropriate length of time.
C. Cross Linking by UV
1. Immediately transfer the membrane to a Stratalinker and expose to UV light (CAUTION! see Hint #2 also see Hint #3).
4. Return the membrane to 2X SSC.
D. Hybridization
1. Wet the membrane with 2X SSC.
2. Place the membrane in Hybridization Buffer and allow to soak for approximately 5 min.
3. Boil 100 μl of radiolabel probe for 5 min then cool on ice for 5 min (see protocol on Hybridization of Nucleic Acids to Membranes).
4. 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 45°C.
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.
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| Tris/Sodium Chloride |
| 1.5 M NaCl
pH 7.4
1 M Tris
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| Chloride/Hydroxide Solution |
| 1.5 M NaCl
0.5 M NaOH
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| Hybridization Buffer |
| 0.5% (w/v) SDS
50% (v/v) Formamide
5X SSC
1X Denhardt solution
0.1 mg/ml denatured Salmon Sperm DNA or Calf Thymus DNA
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| 0.2 M HCl |
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| Denhart Solution |
| 10 g Ficoll 400
Bring final volume to 500 ml using ddH2O
10 g Polyvinylpyrrolidone
10 g Bovine Serum Albumin (fraction V)
Store at -20°C in aliquots
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| SSC (10X) |
| pH 7.2
3 M NaCl
0.3 M Sodium Citrate
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Polyvinyl Pyrolidone
Bovine Serum Albumin (BSA), Fraction V
Ficoll 400
Sodium Chloride
Sodium Hydroxide
SDS
Hydrochloric Acid
Formamide
Sodium Citrate
Tris
DNA, Salmon Sperm
DNA Calf Thymus
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1. The gel should be exposed and the Saran Wrap should be covering the area
2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
3. Make sure the membrane doesn't dry out during the cross linking steps.
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