D:\533571429.doc
1
Northern Procedure
RNA Isolation:
Reference: Chomczynski P, Sacchi N (1987) Analytical Biochemistry 162:156-159.
Solution D (lysis buffer)
Constituents
4 M Guanidine thiocyanate
25 mM Na citrate, pH 7.0
0.5% Na sarcosyl
Preparation
250 g GTC dissolved in 293 ml water at 65°C
add 17.6 ml 0.75 M Na Citrate, pH 7.0
add 26.4 ml 10% (w/v) Na sarcosyl (Na lauroylsarcosine)
sterile filter
(store at RT in sterile bottle 3-4 months)
Solution D+
Preparation
Add 360 l ß-mercaptoethanol (BME) to 50 ml D solution
(store at RT in sterile bottle for 1 month)
Solution (per gram of tissue)
10 ml of D+
990 l 2M NaOAc, pH 4.0
10 ml Phenol
1.8 ml chloroform
*Note: Different volumes of D+/NaOAc/phenol/chloroform can be used but the ratio
must be 1:0.1:1:0.2.
Procedure:
1. Homogenize samples on ice after freezing with liquid nitrogen
2. Add 10 ml/g of D+ to sample in cryotube
3. Homogenize with 3cc syringe and 20 gauge needle OR use Polytron Homogenizer
4. Transfer lysate to eppendorf tube
5. Add 990 l /g NaOAc to each tube, Mix
6. Add 10 ml/g of phenol (for RNA use – different than DNA) and mix briefly
7. Add 1.8 ml/g of chloroform and vortex for 10 seconds
8. Place on ice for 15 minutes
Last printed 2/17/2016 9:32:00 PM
D:\533571429.doc
2
9. Centrifuge at 10K g for 20 min at 4°C
10. Transfer upper layer to eppendorf tube (bottom portion of supernatant has
DNA/protein)
11. Add equal volume of ice cold isopropanol and mix well. Place at –20°C overnight or
at least 1 hour OR at –70°C for less than 15 minutes.
12. Centrifuge at 10K g for 30 minutes
13. Decant supernatant (pellet should be visible – may be loose)
14. Dissolve pellet in 300 l D+. Let hydrate for 5mins – make sure pellet is wet
15. Add 300 l isopropanol and mix. Place at –20°C overnight.
16. Centrifuge at 4°C for 30 minutes on high
17. Decant supernatant and wash pellet in 500 l of 70% EtOH (RNA)
18. Centrifuge at 4°C for 5-10 minutes
19. Decant supernatant and wash pellet in 100l of 70% EtOH (RNA)
20. Centrifuge at 4°C for 5-10 minutes
21. Aspirate supernatant and dry thoroughly (VERY important for next step)
22. Resuspend pellet in appropriate volume of TE, pH 7.2 (100l)
23. Quantitate 1:100 dilution by Abs at 240, 260, and 280 (5ul of above and 495 of TE)
Total RNA (g/l) = (40mg/ml) * (Dilution Factor) * (A260) / 1000
24. Calculate volume for 10g of RNA to load onto the gel.
RNA Formaldehyde Gel for Northern Blotting
alternate receipes)
Solutions:
1M MOPS Buffer pH 7.4:
20.93g/100ml in Depc H20 filter
Adjust to pH 7.4 with 10N NaOH (~10ml)
Store at 4°C
10X MOPS Buffer:
1M MOPS
0.5M EDTA
dH2O
Store at 4°C
Last printed 2/17/2016 9:32:00 PM
100 ml
10 ml
390 ml
(See Appendix for
D:\533571429.doc
3
1.33X MOPS Loading Buffer:
Formamide
Formaldehyde
1M MOPS, pH7.4
0.5M EDTA
20% SDS
1% bromophenol blue/
1% xylene cyanol mix
Glycerol
674 l
216 l
30 l
3 l
5 l
10l
57 l
0.8 % Gel:
Agarose
dH2O
100 ml
0.8 grams
76 ml
heat to dissolve, cool to 60°C and add:
10X MOPS Buffer
10 ml
Formaldehyde
14 ml
Ethidium Bromide can be added to the gel (approx 2l) if desired (decreases
sensitivity)
Running Gel:
1.
2.
3.
4.
5.
6.
7.
Mix 10ug of RNA with equal volume of 1.33X loading buffer in eppendorf tube.
Boil tubes (denature) for 5 minutes and then immediately into ice for 15 minutes.
Run gel at 115 V for approximately 2 hours. Take picture if EtBr was used.
Gel is washed in 1% glycine in 1X MOPS buffer for 30 minutes
Denature in 0.05 M NaOH for 20 minutes
Wash in 10X sodium chloride/sodium citrate (SSC) for 40 minutes.
Gel is now ready for transfer step.
Transfer Setup:
1.
2.
3.
4.
5.
6.
10X SSC solution in tray
Wick (2 sheets of thin filter paper) – Prewet in 10X SSC
Gel – wells up – REMOVE wells.?
Membrane: pre-soak in dH2O
Stack of thick filter paper (5 or 6)
Paper Towels
Allow transfer to run overnight at room temperature.
Last printed 2/17/2016 9:32:00 PM
D:\533571429.doc
4
Cross link and Membrane Staining:
1.
2.
3.
4.
5.
6.
7.
8.
Place membrane on filter paper and place in Stratlinker
Activate autocross link twice
Soak in 5% Acetic Acid for 15 minutes at room temperature
Stain membrane in 0.5M NaOAc pH5.2 and 0.04% Methylene Blue for 5-10
minutes.
Destain with dH2O to remove background.
Mark and photograph membrane for probe reference.
Wash with 2X SSC
Check gel for complete transfer if EtBr was used.
Prehybridization:
1.
2.
3.
Prehybridization solution from Sigma Chemical (kept at –70°C) mixed with equal
volumes of Formamide (Redistilled – the good stuff). Approximately 12 ml is
required.
Membrane is placed on support mesh in hybridization tube
Prehybridization solution is added to the tube and incubated for at least 3 hours at
42°C with agitation (random primer probe).
Random Primer Probe Production:
(Stratagene Prime-It II Random Primer Labeling Kit – kept at –20°C)
PROTOCOL
This protocol is designed to label a DNA probe, at a fixed timepoint, with [-32P]dATP
(3000 Ci/mmol or [-32P]dCTP (3000 Ci/mmol), resulting in maximum specific activity.
NOTE:
If using [-32P]dCTP(6000Ci/mmol) to_qenerate probes with specific
activities >2 x lO9 dpmlmg, the amount of label per reaction should be 10l
(or 100 Ci), and the volume of water should be reduced by 5l/
To determine the optimal labeling time for a specific DNA template, see Appendix II:
Measurement of Probe Specific Activity.
1. Add the following components to the bottom of a clean microcentrifuge tube:
Sample:
25 ng (1-23 l) of DNA template to be labeled.
0-23 l of high quality H20. Use this to bring final solution volume to 34 l.
10 l random oligonucleotide primers.
Total reaction volume should be 34 ul at this point.
Last printed 2/17/2016 9:32:00 PM
D:\533571429.doc
5
2. Heat the reaction tubes in a boiling water bath for 5 minutes and then centrifuge briefly
at room temperature to collect the liquid, which may have condensed an the cap of the
tubes. If the DNA sample is in LMT agarose, after removal from the boiling water bath
and centrifugation, place the reaction tube at 37° and then proceed to step 3. If the DNA
sample is in aqueous solution, leave the reaction tube at room temperature and then
proceed to step 3.
3. Add the following reagents to the reaction tubes:
a.
10 l of 5x primer buffer
b.
5 l of labeled nucleotide: Use either [-32P]dCTP at 3000 CVmmole of
[-32P]dATP at 3000 Ci/mmol. Mix the contents of the tube thoroughly
with your pipet tip.
c.
1 l Exo(-) Klenow enzyme
4. Mix the reaction components thoroughly with your pipet tip.
5. Incubate the reactions at 37-40°C for 2-1 0 minutes.
6. Following the incubation period, stop the reaction by adding 2 l of Stop Mix.
NucTrap Probe Purification Columns:
1.
2.
3.
4.
5.
6.
7.
8.
Prewet the NucTrap push column with 75l of 1X STE (100 mM NaCl; 20 mM
Tris-HCl pH7.5; 10mM EDTA).
Extend the syringe plunger and attach syringe to column. Push slowly to the end
when a small drop of STE should emerge (columns can be bad). Use within 5-10
minutes.
Add 1X STE to the probe mix to bring the final volume to 70 l.
Place a microcentrifuge into the holder. Insert the prewet column into the holder.
Using beta shield device load the sample to the top of the NucTrap push column.
Extend the plunger on the syringe. Attach syringe to column and slowly push the
plunger to expel the probe.
Wash the column with an additional 70l of 1XSTE and collect in the
microcentrifuge tube.
Remove the column and probe from the beta shield device.
Probe Activity:
1.
2.
Test activity by pippeting 1 l of probe into a scintillation vial and read cpm on
beta counter.
Calculate dilution for probe in hybridization solution. A final activity of 1x10 6
per ml is desired for 12ml of hybridization solution.
Last printed 2/17/2016 9:32:00 PM
D:\533571429.doc
6
Hybridization:
1.
2.
3.
4.
5.
Hybridization solution from Sigma Chemical mixed with equal volumes of
Formamide. Approximately 12 ml is required.
Add probe at a final concentration of 1x106 counts per ml
Boil mixture for 10 min in 50 ml tube. Immediately place on ice for 5 min.
Remove prehybridization solution from tube and replace with above solution.
Hybridize at 42°C overnight (65°C for 5’ end labeling)
Wash Membrane and Autoradiography:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Pour off hybridization solution and store at –70°C (dependent on ½ life of P32)
Wash membrane in tube with 2X SSC (approximately 50 ml) – quick wash
Remove membrane and place in plastic tray
Add approximately 100 ml of 2xSSC/0.1% SDS and incubate with agitation for 5
min at RT - Repeat
Replace solution with 0.2x SSC/0.1% SDS and incubate with agitation for 5 min
at RT – Repeat.
If desired carry out two further washes using prewarmed (42°C) 0.2x SSC/0.1%
SDS for 15 min each at 42°C (moderate stringency wash)
Rinse blot in 2x SSC (quick wash)
Drain blot and wrap in plastic for exposure to film.
Place blot in film cassette and place film on top of plastic/blot
Expose at –80°C overnight/days depending on signal strength.
Remove film and develop.
Membrane Stripping (Ref Current Protocols):
- Membranes can be safely stripped for up 3 times (PG)
1.
Stripping Solution: 1% SDS, 0.1X SSC, and 40 mM Tris-Cl pH 7.5-7.8
2.
To remove probes at 80°C: Place membrane in a dish containing stripping
solution without formamide. Place dish in preheated waterbath (80°C for 5
minutes)
3.
Pour off solution and then repeat 3-4 times.
4.
Continue with Prehybridization steps.
Last printed 2/17/2016 9:32:00 PM
D:\533571429.doc
7
Alternate Methods/Recipes:
RNA Denaturing Solution (P. Goldspink):
1.
2.
3.
20ul of 100% Formamide
7ul of Formaldehyde (37%)
2ul of 20X SSC
Use 29 ul of solution per 10 ul of RNA solution. Incubate samples at 68°C for 20
minutes. Place on ice and add DNA loading dye. Load samples (preferably use large
combs).
RNA Gel (P. Goldspink):
1.
69.6 ml dH2O
2.
0.96 g Agarose
HEAT to dissolve
3.
8 ml 10X MOPS Buffer
4.
2.4 ml Formaldehyde (37%)
5.
Ethidium Bromide
Last printed 2/17/2016 9:32:00 PM