SOUTHERN BLOTTING PROTOCOL
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SOLUTIONS
Lysis Buffer
0.1 M Tris, pH 8.0
0.2 M NaCl
5 mM EDTA
0.2% w/v SDS
200 m g/ml proteinase K
TE Buffer
10 mM Tris-Cl, pH 7.4
1 mM EDTA, pH
Denature
Neutralize
20X SSC
Membrane Wash
WHITE LAB � SOUTHERN BLOTTING PROTOCOL
GENOMIC DNA EXTRACTION
1. Cut mouse tails, place in tubes, and add 0.5 ml of fresh lysis buffer [0.1M Tris, pH 8.0; 0.2M NaCl; 5mM EDTA; 0.2% w/v SDS; 200 m g/ml proteinase K] to each tube
2. Digest overnight in 55ºC waterbath
3. Spin tubes for 20 minutes at full speed to pellet tail debris
4. Remove supernatant (avoid hair & debris) to 24-well plates containing 0.75 isopropanol
Use wide-orifice tips to avoid shearing the DNA.
5. Leave plates on rocker for 40+ minutes, until DNA precipitates are visible
6. Use wide-orifice tips to remove precipitated DNA to tubes containing 0.5 ml 70% EtOH
7. Spin tubes for 10 minutes at full speed
8. Use gel loading tips to aspirate pellet � make sure pellet is dry
9. Add 100-250 m l TE to each tube
10. Incubate overnight in 55ºC waterbath
11. Store at 4ºC
DIGESTION OF GENOMIC DNA
12. Aliquot 15 m l of genomic DNA into fresh labeled tubes
13. Make up appropriate restriction enzyme digestion and add to tubes
14. Incubate overnight in 37ºC waterbath
GEL ELECTROPHORESIS
15. Run samples on 0.8% agarose gels at ~ 150V for approximately 3-4 hours
16. Denature gels for 45 minutes on rocker in denature solution [1.5M NaCl; 0.5M NaOH]
17. Neutralize gels for 45-60+ minutes on rocker in neutralizing solution [1M Tris; 1.5M NaCl; pH 7.4]
18. Set up transfer using 10X SSC and Hybond � transfer overnight
19. Mark well positions on membranes
20. Cross-link the DNA to the membranes using the UV crosslinker
21. Stain for 5 minutes in water with a little EtBr
22. De-stain in water for 20 minutes and check under UV to verify transfer
HYBRIDIZATION AND PROBE SYNTHESIS
23. Prehyb [30 ml of 20X SSC; 2.5 ml of 20% SDS; 50 ml of formamide; 5 ml of 100X Denhardts (or 10 ml of 50X); 12.5 ml of H2O; 1 ml of herring sperm] for 4-6 hours in 42ºC shaking waterbath in covered containers � place 1 ml of herring sperm DNA in heatblock for 5 minutes, transfer to ice for 5 minutes, then add to prehyb solution
Need approximately 100 ml of prehyb to prehyb and probe 4 membranes, 50 for prehyb and 20-30 for hybridization
24. Make probe according to NEB nick translation kit and purify with Nick columns or Qiagen Nucleotide Removal kit
25. Place probe on heatblock for 5 minutes; transfer to ice for 5 minutes
26. Add probe to 20-30 ml of prehyb mixture; mix thoroughly
27. Add membranes to container with mixture; make sure they aren’t sticking together
28. Hybridize overnight at 42ºC in shaking waterbath
29. Add membrane wash [2X SSC; 0.5% SDS] to fresh containers
30. Remove membranes from probe solution and add to wash containers
31. Wash twice for 20 minutes in 42ºC shaking water bath
32. Let membranes dry on Whatman paper
33. Wrap in Saran Wrap and expose overnight on autorad cassette or PhosphorImaging cassette
34. Decant probe into waste tube or save in falcon tube
35. Develop film