Genomic DNA Digestion with Restriction Enzyme
For movo1 genotyping, we use Pvu II to cut.
(follow DNA isolation protocol for tail DNA)
Bigger gel apparatus Smaller gel apparatus
Tail DNA 10 μl 6.5 μl
10X NEB R#2 3 μl 2 μl
Pvu II (10U/ μl) 3 μl 2 μl
dd H2O 14 μl 9.5 μl
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Total volume 30 μl 20 μl
1. If digestion complete, add 6X loading dye to digested DNA, mix and incubate at 65C for 5 min.
2. Load samples to a big 0.7% agrose gel (without any EtBr in gel or buffer), set 150-200 V for 5-10 min, then change to 30-50 V for rest of the time.
3. Stain the gel in 2.5 g/ml EtBr for 15 min, take picture.
4. Destain in water for 30 min on shaker.
5. Depurinate in 0.25M HCl for 15 min on shaker. (gel color change from blue to yellow)
6. Denature in 0.5 N NaOH and 1M NaCl for 30 min on shaker. (from yellow back to blue)
7. Neutralize in 0.5 M Tris(pH 8.0) and 1.5 M NaCl for 30 min on shaker.
8. Set up blotting overnight.
9. Rinse the blot in 10*SSC quickly, set it onto the filter paper, UV-crosslink it
10. Wrap it and store at 4 C. Or go on to the hybridization if fresh 32P available.
Hybridize for Southern Blot and Northern Blot
1. Pre-heat expression solution at 68C until resolved.
2. Incubate the blot in 6*SSC at the same time.
3. Prehybridize blot in 10 ml expression solution for at least 1h at 60C
4. Make probe: Mix DNA probe fragment (25-100 ng) (see note 4) and mili-Q water to 11l.
Boil at 100C for 10 minutes, quickly transfer onto ice, set on ice for 5 min.
Add 5 l 32P-dCTP, then 4l High-Primer (stored at –20 °C small freezer) .
Incubate at 37 C for 10 min. (may extend to 15-20 min if 32P is not very fresh).
Pass the nick-column and check radioactivity.
Save half of the labeled-probe (~ 200 μl) at –80 °C freezer for later use.
4. Denature the rest half probe: Boil at 100C for 5 min, quickly transfer onto ice, set on ice for 5 min.
5. Pour off pre-hybridized expression solution, add 6ml new expression solution with labeled probe into the hybridization tube.
6. Hybridize in the oven for overnight at 60C
7. Wash the blot: ( see Note 5):
Wash 1— 30 min, 3 times at 50 C.
Wash 2— 50 min, 2 times at 50C
8. Wash briefly in 0.1*SSC. Check cpm and wrap it.
9. Expose at -80C, (the time is depending on cpm count on washed blot.), develop the films 1 to 2 days later.
Strip the Probe Off Southern Blot
1. Boil 0.5% SDS solution in a beaker (microwave).
2. Soak the blot into hot SDS solution, incubate for 10 min, shaking frequently.
3. Transfer the blot to 2*SSC, incubate for 5 min at room temperature.
4. Wrap and store at 4C.
NOTES
Note 1: TNES buffer For 50 ml:
50 mM Tris-HCl (pH7.4) 1 M Tris-HCl (pH7.4) —– 2.5 ml
100 mM EDTA (pH8.0) 0.5 M EDTA (pH8.0) —– 10 ml
400 mM NaCl 5 M NaCl —– 4 ml
0.5% SDS 10 % SDS —– 2.5 ml
dd H2O —– 31 ml
Note 2: Proteinase K (20mg/ml)
—– Proteinase K powder is shipped and stored at -20°C freezer.
—– Making stock solution: add 2.5 ml dd H2O to the bottle(50 mg/each bottle) and mix by inverting, then aliquot into small amount vials, ( usually 50 μl) and store in the Proteinase K stock solution box in the -20°C freezer.
Note 3: 6M saturated NaCl
173.5g NaCl/500 ml dd H2O, 65°C heat to dissolve.
Note 4: DNA probe fragment for movo1 alleles
—– Mini-prep DNA from plasmid of pCMO-N1pstrc. (In Glycerol stock box I-75, or you could check the lab resources folder)
—– Cut with EcoRI and Sal I:
Plasmid DNA ~ 5.0 μg
10 X Gibco R#2 buffer 10 μl
Sal I 6 μl
EcoRI 4 μl
dd H2O 75 μl
Total volume 100 μl
—– Gel purify the 1.5 kb fragment, quantitate and DNA concentration and store at –20 °C.
Note 5: Post-hybridization washing solutions:
Wash 1— 2*SSC, 0.05% SDS
Wash 2— 0.1*SSC, 0.1% SDS