WHITE LAB SOUTHERN BLOTTING PROTOCOL

 

SOLUTIONS

 

Lysis Buffer for tails

 

0.1 M Tris, pH 8.0

0.2 M NaCl

5 ml EDTA

H2O

0.2% w/v SDS

autoclave

200 mg/ml

 

TE Buffer, pH 7.4

10 mM Tris-Cl, pH 7.4

1 mM EDTA

 

Denature

1.5 M NaCl

0.5 M NaOH

 

Neutralize, pH 7.4

 

1.5 M NaCl

1 M Tris

 

20X SSC, pH 7.0

 

3 M NaCl

0.3 M Sodium Citrate

 

50X Denhardts, 500 ml

 

5 g ficoll

5 g polyvinylpyrrolidon (PVP)

5 g BSA

H2O to 500 ml

filter mixture

 

Prehybridization buffer

 

6X SSC

50% formamide

12.5% water

5X Denhardts

0.5% SDS

 

Membrane Wash

 

0.5% SDS

2X SSC

 

Stripping Solution

0.1% SDS

0.1 X SSC

 

DNA Dye

 

7.5 ml 40X TAE

18.75 ml 80% glycerol

7.5 ml sterile H2O

0.03 g bromophenol blue

0.03 g xylene cyanol

 

 


WHITE LAB SOUTHERN BLOTTING PROTOCOL

 

GENOMIC DNA EXTRACTION

 

1. Cut tails and mark ears as follows:

no cuts: 1

left ear cut: 2

right ear cut: 3

both ears cut: 4

short tail: 5

2. Place tails in Eppendorf tubes and add 0.5 ml of fresh lysis buffer to each tube

(Fresh = add proteinase K just before using)

3. Digest overnight in 55C waterbath

 

4. Spin tubes for 20 minutes at full speed to pellet tail debris

5. Use wide-orifice tips to place supernatant in 24-well plates with 0.75 ml isopropanol

6. Place plates on rocker for 40+ minutes, until DNA precipitates are visible

7. Use wide-orifice tips to remove precipitated DNA to tubes containing 0.5 ml 70% EtOH

8. Spin tubes for 10 minutes at full speed

9. Use gel loading tips to aspirate pellet make sure pellet is dry

10. Add 100-250ml TE to each tube (for more concentrated DNA, add 100 ml TE)

11. Incubate overnight in 55C waterbath

 

12. Store at 4C

 

DIGESTION OF GENOMIC DNA

 

13. Aliquot 15ml of genomic DNA into fresh labeled tubes

14. Make up appropriate digestion mixture:

(29 ml H2O, 5 ml enzyme, 5 ml enzyme buffer, 0.5 ml 100 X BSA)

IRS-1: BamH1 with BamH1 buffer

IRS-2: Spe1 with buffer 2

STF: EcoR1 with EcoR1 buffer

IGF: HincII with buffer 3

IMM/TG: BamH1 with BamH1 buffer

15. Add 40ml of digestion mixture to each tube

16. Incubate overnight in 37C waterbath

 

GEL ELECTROPHORESIS

 

17. Make 0.8% agarose gels

18. Add 5ml of DNA dye to tubes containing digested DNA

19. Spin briefly to mix

20. Load entire sample into wells

21. Run at ~150V for approximately 3-4 hours

22. Cut gels and place in numbered containers add Denature solution

23. After 45 minutes on rocker, pour off Denature and add Neutralize solution

24. Set up transfer, using 10X SSC and Hybond transfer overnight

 

 

25. Mark well positions on membranes

26. Cross-link the DNA to the membranes (UV crosslinker is in the Kahn lab)

27. Stain in water+EtBr for 5 minutes

28. De-stain in water for 20 minutes

 

HYBRIDIZATION AND PROBE SYNTHESIS

 

29. Add prehybridization mixture (50 ml per 4 membranes) to covered containers

30. Prehyb for 4-6 hours in 42C shaking waterbath

31. Make probe according to NEB nick translation kit

32. Label ~5 ml of probe DNA (exact amount to label is determined by concentration)

33. Add appropriate dNTPs, 5 ml of 32-P dCTP, and 1 ml Klenow

34. Incubate in plexiglass radiation box for hour in 37C incubator

35. Add 1 ml of Klenow and place in incubator for another hour

36. Use Nick column or Qiagen kit to purify probe

37. Add 20-40 ml of prehyb solution to fresh, covered containers

38. Boil probe on radiation hotblock for 5 minutes; transfer to ice for 5 minutes

39. Add to container with prehyb and swirl to mix thoroughly

40. Carefully transfer membranes from prehyb containers to probe containers

41. Mix to ensure membranes arent sticking to one another

42. Hybridize overnight at 42C in shaking waterbath

 

[If you are re-using a probe, replace steps 37-39 with the following:

37*. Boil falcon tube containing used probe for 15 minutes; place on ice for 15 minutes

38*. Add to new container.]

 

43. Add membrane wash to fresh tupperware

44. Remove membranes from probe solution and add to wash tupperware

45. Place wash tupperware in 42C shaking bath for 20 minutes

46. Dump out wash and add more for another 20 minutes

47. Remove membranes from wash and place on Whatman paper to dry

48. When dry, place in SaranWrap

49. Expose overnight on autorad cassette or PhosphorImaging cassette.

50. Decant probe into waste tube or save in falcon tube (in plexiglass box in refrigerator)

 

51. Develop film and score