Northern Blot
From CCGB
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Northern Bolt
-- by Hao Wang
Preparation of samples:
1. Aspirate off media from cells. Overlay with TRIzol (1 ml for 6 well plate and 10 cm plate or 2 ml for 15 cm plate).
2. Scrape cells, homogenize by pipetting, and place in sterile Eppendorf tube (~1 ml each tube). Incubate @ r.t. for 5 in.
3. Add 200μl chloroform per 1 ml TRIzol. Mix gently by inverting ~ 25 times. Incubate at r.t. for 30 min.
4. Centrifuge at 12,000 g for 15 in at 4°C.
5. Remove top aqueous layer to new tube. Add 500 μl isopropanol per 1 ml TRIzol. Incubate @ r.t. for 10 min.
6. Centrifuge @ 12,000 g for 10 min @ 4 °C.
7. Remove supernatant. Wash pellet with 1 ml 75% DEPC Ethanol (do not resuspend pellet).
8. Centrifuge @ 7,500 g for 5 min @ 4°C.
9. Remove supernatnant or add 70% DEPC Ethanol and keep @ -80°. Let air-dry. Then resuspend in 10-20 μl 100% formamide.
Preparation of Agarose Gel:
1. Wash gel apparatus with ddH2O. Soak in sterile 75% EtOH for 30 min. Then rinse with sterile H2O.
2. Cast gel: 1% agarose gel:
In sterile bottle, add: 52.5 ml 1× MOPS buffer + 0.625g agarose.
Microwave until completely dissolved. Let cool to 65°C.
Add 10 ml of 37% formaldehyde in hood to final [ ] of 6%.
Add 5 μl EtBr and mix.
Pour and let dry 1 hr in the hood.
3. Prepare samples to load:
Read absorbance at 260/280 nm to calculate concentration. Use 1 μl RNA for OD260 (1 μl formamide as background).
Add 20 μg RNA up to 8.8 μl total volume with formamide.
Add 3.2 μl formaldehyde + 4μl 5× MOPS buffer.
Incubate at 55°C for 15 min.
Add 4 μl loading buffer.
4. Pre-run the gel for 5 min in 1 × MOPS buffer to get rid of any RNAse in well.
5. Load gel and run at 100V until bromophenol blue dye is ~ 1 cm from edge.
Preparation of Agarose Gel (Alternative protocol)
1. To prepare FA gel (1.2%) agarose) of size 15×20×0.65cm, mix
2. Heat the mixture to melt agarose.
3. Cool to 65°C in a water bath.
4. Add 36 ml of 37% (12.3M) formaldehyde and 10 μl of a 10mg/ml ethidium bromide stock solution.
5. Mix thoroughly and pour onto gel support. Prior to running the gel, equilibrate in 1×FA gel buffer for at least 30min.
Transfer of RNA from gel to membrane:
1. Take picture of gel with rulers before transferring to membrane.
2. Clean big protein gel glass plate with EtOH. Air-dry. Place on Saran wrap.
3. Prewet Nylon-Plus (positively charged) membrane with sterile ddH2O and then soak in 20 × SSC.
4. Pour 20 × SSC on glass plate. Place 2 pieces of clean mat paper on galss and remove any air bubbles by rolling over with plastic pipet.
5. Place gel with wells facing down. Get rid of any air bubbles and then place membrane on gel.
6. Overlay with more Saran wrap and then fold corners in so that 20 × SSC remains between the two pieces of Saran wrap.
7. Remove Saran wrap where the gel and membrane are with a razor.
8. Place similar size mat paper on top. Then place at least 3 inches of paper towels on top.
9. Place plastic container and weight on top to press down. Leave O/N and change paper towels when necessary.
Preparation of Radiolabelled Probes:
1. Denautre 25-50ng DNA probe by heating @ 95-100°C for 2 min. Place on ice immediately.
2. Then add: (Amersham Ready-To-Go DNA labeling beads (-dCTP))
Denatured DNA <=45 μl
[α-32P]dCTP (3000 Ci/mmol) 5μl
Distilled water to total of 50μl
3. Mix by gentlypipetting up and down several times or by gentle vortexing. If bubbles appear they may be removed by a pulse centrifugation.
4. Incubate @ 37°C for 15 min to 1 hr.
5. Quick Spin TE column purify:
Resuspend column and make homogenous. Make sure there are no air bubbles.
Remove top and bottom caps and let drain.
Spin 2000 rpm for 2 min.
Change collection tube to new tube and then add above DNA sample to center of column bed.
Spin 2000 rpm for 4 min.
Collection tube contains radiolabelled probe.
Store at -20°C or use immediately by boiling 5 min and keep on ice.
Hybridization of Blot:
Day1:
1. Remove gel and membrane together from transfer apparatus. Then mark the wells with a pencil on the membrane.
2. Place membrane in 0.05 N NaOH. Let soak for 5 min with RNA side up.
3. Then soak in 2 × SSC for 2 min.
4. Wash hybridization chamber with sterile H2O. then place membrane inside chamber-RNA side facing up and removing any air bubbles underneath.
5. Add 6 ml ULTRAhyb hybridization solution (Ambion). Rotate chamber @ 42°C for 1 hr.
6. Pour off solution and add 6 ml fresh solution. Place rediolabelled probe directly into solution and rotate @ 42°C O/N. Remember to boil the probe for 5 min before use.
Day 2:
1. Prepare 200 ml wash solution: 0.1 × SSC + 0.1% SDS in sterile H2O.
2. Pour out probe/hybridization solution in liquid radioactive waste.
3. Rinse with 5-10 ml wash solution and pour into liquid radioactive waste.
4. Wash 3 × 10 min @ 55°C.
5. Remove wash solution. Let drip but don’t let blot dry out!
6. Wrap in saran wrap and then place on mat paper and warp again.
7. Expose to phosphoimager or cassette with BioMax film @ -80°C.
Stripping the Probe:
2 liter of 2 L 1.5 L 10 mM Tris ph7.5 1 M 20 ml 15 ml
0.1 × SSC 20 × 10 ml 7.5 ml
1 mM EDTA 0.5 M 4 ml 3 ml
0.5% SDS 10% 100 ml 75 ml
H2O 1866 ml 1400 ml
Add small stir bars
1. Heat to 95°C-100°C.
2. Turn off heat, put in membranes.
3. Let sit for 15 min. While the membrane is in, heat a second 1.5 L.
4. Pour off 1st stripping buffer and add 2nd for 15 min.
5. Remove. Wash with 0.2 × SSC 5 min twice.
6. Dry or reprobe.
Composition of FA gel buffers
10×FA Gel Buffer
200 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (free acid)
50 mM sodium acetate
10 mM EDTA
pH to 7.0 with NaOH
1×FA Gel Running Buffer
100 ml 10×FA gel buffer
20 ml 37% (12.3M) formaldehyde
880 ml RNase-free water
5×RNA Loading Buffer
16 μl saturated aqueous bromophenol blue solution
80 μl 500 mM EDTA, pH8.0
720μl 37%(12.3M) formaldehyde
2 ml 100% glycerol
3084 μl formamide
4 ml 10× FA gel buffer
RNase-free water to 10 ml
Stability: Approximately 3 months at 4°C
