0.1 M Tris, pH 8.0
0.2 M NaCl
5 ml EDTA
H2O
0.2% w/v SDS
autoclave
200 mg/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
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 55ºC 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 55ºC waterbath
12. Store at 4ºC
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 37ºC 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 42ºC 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 ½ hour in 37ºC incubator
35. Add 1 ml of Klenow and place in
incubator for another ½ 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 aren’t sticking
to one another
42. Hybridize overnight at 42ºC 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 42ºC 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