1. Rinse and then wash the membrane under high stringency conditions:
a. 0.1% SDS, 2* SSC for 5min at RT *2
wash I: 10% SDS 1ml; 20* SSC 10ml; DEPC H2O 89 ml b. 0.1% SDS, 0.1* SSC for 15min at 55℃ *2
wash II: 10% SDS 1ml; 20* SSC 0.5ml; DEPC H2O 98.5 ml
2. Prepare 50 ml of blotting buffer 1:10 Blocking reagent in maleic acid buffer. 3. To adjust the PH, soak the membrane in washing buffer for 3 min. 4. Incubate membrane for 1h at RT in blocking buffer.
5. Incubate membrane for 1.5h at RT in antibody solution (anti-DIG antibody #4 diluted 1:10000 in blocking buffer) on shaker.
6. Wash membrane with washing buffer 2*15 min on shaker. 7. Equilibrate for 3 min in detection buffer.
8. Take the membrane out of the detection buffer, and put into a plastic envelope add CSPD and make it well-proportioned by lifting up one side of the envelope up and down. Then incubate at RT for 5 min.
9. Put the membrane in a new envelope; incubate at 37℃ for 30 min. 10. X-ray film exposure. Stripping membrane
1. PH 7.5 10mM Tris-HCl, 1mM EDTA, 0.1%SDS 95℃ for 5 min *2 2. Membrane keep in 2*SSC
[10] Extraction of DNA from Tissue
1. Grind the tissue to powder under liquid nitrogen using a mortar and pestle. Collect 300mg of powder into a sterile tube. 2. Add 10 volumes (3ml) of extraction buffer. 3. Add 30μl Proteinase K (20 mg/ml). 4. Incubate at 55℃ 3h - overnight. 5. Cool down to RT.
6. Add 7.5 μl 10mg/ml RNase A.
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7. Incubate at 37℃ for about 1h.
8. Extract with equal volume (3ml) of phenol (酚) saturated (PH 8.0). Mix at RT for 20 min on a shaker.
9. Centrifuge at 3,500* g for 10min at 4℃.
10. Transfer the viscous (黏性的) aqueous phase into a new tube. Repeat once from step 8. 11. Add equal volume (3ml) of PCI. Mix at RT for 20 min on the shaker. 12. Centrifuge at 3,500*g for 10 min at 4℃.
13. Transfer the viscous aqueous phase into a new tube. 14. Prepare for two tubes of 70% ethanol.
15. Slowly add 2 volumes of 100% ethanol into the sample tube (step 11), to produce two phases.
16. Carefully mix the two phases with a glass rod and spool DNA onto it. 17. Put the glass rod into 70% ethanol. Rinse twice.
18. Air-dry at RT. Then dissolve in 0.5 ml of TE buffer overnight at 4℃.
19. Measure the OD at 260 nm and 280 nm to get the concentration of the DNA solution. DNA (mg/ml) =A260 * 0.05* dilution factor. 20. Store the genomic DNA at 4℃ for about 2-3 months.
[11] Southern blotting using DIG high primer labeling kit
First day
1. Prepare 1% agarose/TAE gel. Make sure gels are filled to the top. 2. prepare DNA sample.
3. Run the gel in 1* TAE, following by 25-50 V/25 cm (~4-8 h, lower voltage will separate the samples better).
4. While the gel is running, prepare the blotting apparatus:
a) fill square Pyrex dish with reused 10* ssc . Wrap glass plate with blotting paper
and place over the dish, allowing sides of blotting paper to drop down and soak up the buffer;
b) Cut three pieces of blotting paper and one piece of nylon membrane to the same
size as the gel;
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c) Cut enough paper towels to the same size as the gel, to make a stack 10-13 cm
tall.
5. Depurination (Optional for DNA >10 kb): Submerge the gel in 250 mM HCl for depurination the DNA until the bromophenol blue marker changes from blue to yellow (human <10 min, plant <20 min).
6. Denaturation: Soak in Denaturation Buffer 0.5 M NaOH/1.5 M NaCl solution for 2×15 min.
7. Neutralization: Neutralize the gel by soaking in Neutralization Buffer 0.5 M Tris (pH 7.5)/1.5 M NaCl for 2×15 min.
8. Equilibrate the gel by soaking in 10* ssc for 10 min.
9. While the gel is soaking in ssc, place the membrane in dH2O for ~5 min and then replace the water with fresh 10*ssc
10. Pour fresh 10*ssc on top of the blotting apparatus and remove all air bubbles from the blotting paper e.g. by rolling a glass pipette over the surface. Make sire the plate is filled ~3/4 with 10*ssc
11. Plate the gel on the covered plate upside down; i.e. the well holes face down and the smooth side of the gel faces up. Remove all air bubbles between gel and plate, wet the gel with 10* ssc.
12. Place the membrane on the top of the gel and remove all bubbles. Cover with three pieces of the blotting paper, also making sure that no bubbles are present.
13. Cover entire apparatus with plastic-wrap and using a razor carefully cut a hole in the plastic around the gel. Remove the cut plastic and seal the apparatus with the remaining plastic to prevent evaporation.
14. Place ~7.5-9cm of the paper towels on top of the gel. Lay another glass on the gel and add sufficient weight on top to compress the towels, but not crush the gel. 15. Allow to running overnight, replacing the wet towels as necessary.
Second day
7. Remove paper towels and blotting paper. Mark the position of the wells on the membrane using a pencil. Save SSC buffer for reuse.
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8. Place membrane on a clean paper and incubate in 37℃ to dry. Immobilize the nucleic acids to the membrane by UV crosslink(120mJ/cm2) for ~1min. Membrane can now be stored at RT or 4℃(better) with no degradation.
9. Place the membrane in a small plastic container and prehybridize the membrane for 1h in DIG Easy Hyb at 42℃ in a water bath.
10. Just before prehybridization is completed, denature the probes
a. add 40 ul DIG Easy Hyb to labeled probes, vortex and spin down. b. Incubate at 95℃ for 5min and chill on ice water(1 min). 11. Add probes to DIG Easy Hyb buffer and mix gently.
12. Place membrane in a plastic envelope, add probes and heat seal. Place the envelope in a small plastic dish with enough distilled H2O to cover the bag. Incubate at 42℃ overnight.
Third Day
11. Rinse and then wash the membrane under high stringency conditions:
a. 0.1% SDS, 2* SSC for 5min at room temperature ×2 wash I: 10% SDS 1ml; 20* SSC 10ml; SDW 89 ml b. 0.1% SDS, 0.1* SSC for 15min at 55 ℃ ×2
wash II: 10% SDS 1ml; 20* SSC 0.5ml; SDW 98.5 ml (Wash II may be adjusted empirically)
12. Prepare 50 ml of blotting buffer 1:10 Blocking reagent in maleic acid buffer. 13. To adjust the PH, soak the membrane in washing buffer for 3 min. 14. Incubate membrane for 1h at RT in blocking buffer.
15. Incubate membrane for 1.5h at RT in antibody solution(anti-DIG antibody #4 diluted 1:10000 in blocking buffer) on shaker.
16. Wash membrane with washing buffer 2*15 min on shaker. 17. Equilibrate for 3 min in detection buffer.
18. Take the membrane out of the detection buffer, and put into a plastic envelope add and make it well-proportioned by lifting up one side of the envelope up and down. Then incubate at RT for 5 min.
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