NORTHERN PROTOCOL
RNA HARVESTING & SAMPLE PREPARATION
(NOTE: If concerned about RNAse contamination of pipettors, the shafts can be soaked
in chloroform)
1. Remove media
2. Add 500-700µL RNAzolB (mireya's fridge) per 60 mm dish (500 or less for well of a
6-well). Scrape cells off dish into Eppy.
3. Add 1/10 V chloroform
4. Mix vigorously (shake by hand), keep on ice 5'
5. Spin in cold room 15', max
6. keep aqueous phase (top) - the bottom phase is purple. Make sure not to take the white
junk along, use a P200.
7. Add equal V isopropanol, mix
8. keep on ice 30'
9. spin max, 15' cold room
10. wash in 70% EtoH STORE HERE AT -20 IF YOU WILL NOT USE RIGHT
AWAY
11. spin 15', cool room max speed
12. resuspend in 20µL DEPC ddH2O to spec or 20µL loading buffer to use
13. If you spec, then reppt RNA with 1/10V NaOAc pH 5.2, 2X V 100% EtOH on ice
30', spin at 4° 30', wash in 70% EtOH as above.
14. For spec, 1 OD = 40µg/mL
15. Before loading, heat samples at 65°C for 10' and cool on ice.
ALTERNATIVE SAMPLE PREPARATION
Sample Buffer (2X Denaturing Buffer):
50 ul formamide
18 ul 37% formaldehyde (~ 2.2 M)
10 ul 10X MOPS buffer
Loading Buffer (Add 1 ul EtBr to 100 ul loading buffer)
10 mM EDTA Ph 8.0
0.25% (w/v) bromphenol blue
0.25% (w/v) xylene cyanol
50% (v/v) glycerol
RNA sample preparation:
< 8 ul RNA sample + 10 ul sample buffer
Heat @ 65 C for 10 min (don’t forget to heat the samples)
Cool on ice immediately
Add 2 ul loading buffer (w/ EtBr)
Load on gel
FORMALDEHYDE GEL
1. Treat 250mL flask and a stir bar, gel box and combs with Rnase Zap and rinse with
DEPC water
3. For 1.2% gel: 1.08g agarose, 65 mL DEPC ddH2O. Heat in microwave to dissolve
4. Add stir bar and stir in hood until 65°C (can hold in hand)
5. Add 9mL 10X MOPS, 16mL 37% formaldehyde (jug under the hood near tc room).
6. Swirl to mix and pour into gel box in the hood. Not too thick!
7. Leave in hood until cool, then can move to bench.
8. Running buffer is 1X MOPS (DEPC ddH2O, about 600mL for medium box)
9. Load samples, load dye in the first lane for tracking just use DNA SB in loading buffer
(or you can leave this out if you are using the alternative sample preparation).
Record order!
10. Run about 70-100V (takes 2.5 -3 hours for dye to reach the bottom at 70V)
11. Clean a plastic container very well with Rnase Zap and ddH2O. Wash gel 6X fresh
ddH2O each for 5 min.
12. Wash gel 20X SSC for 30'.
13. Assemble turbo blotter (Jay's bench, paper in cabinet near Wen's bench) according to
the manufacturer's directions. (wash with Rnase Zap and ddH2O then dry, then 20
#4s, 3 #2s, 2 #2s soaked in 20X SSC, the Hybond nylon membrane cut to gel size,
the gel face up NO BUBBLES, then line the area around the gel with parafilm so the
transfer won't short circuit, then more #2s in 20X SSC, then the wick, with about
100mL 2X SSC in the buffer chamber.) Make sure to line Hybond nylon with
parafilm so you do not short circuit transfer.
14. Wrap turbo blotter in saran wrap and transfer O/N.
BLOTTING
1. Next am, take down transfer. Mark position of lanes at the top with pencil. Cut upper
left corner.
2. Wet only the back side of blot in 2X SSC by floating it on the top of a container of
SSC
3. Place on saran wrap face up and U/V crosslink 4' (room with phosphorimager).
4. Rinse in 2X SSC
5. 5' in 1N acetic acid (reuse this solution)
6. 5' in methylene blue stain (reuse this solution).
7. destain with ddH2O from the spigot.
8. Wrap blot and xerox to document rRNA bands. Mark position with pencil in the dye
lane.
9. Store at -80 until ready to probe.
10. To ready for probe, prehyb. 90' in 15 mL hybridization solution at 65°C in warm
room rotator.
PROBE
1. PCR or digest probe sequence 500-1000 bp, you'll need 25-50ng per probe
2. Mix 25-50ng DNA into TE for a final volume of 45µL
3. Denature 95-100°C for 5 minutes.
4. Cool on ice for 2 minutes
5. Centrifuge briefly
6. Get a "Ready to Go" probe tube from Mireya's RNAse free cabinet (Pharmacia)
7. Make sure white pellet is at the bottom of the tube.
8. Add the 45µL denatured DNA and dissolve pellet. Pipette carefully to avoid bubbles.
9. Add 5µL fresh (preferably) 32P-dCTP (NEN, Easytides, 50µCi)
10. dCTP is green, pipette to make uniform solution.
11. Place in radioactive box in warm room (37°) for 5-30 mintues (I've been doing 30).
12. Prepare column (from Bob’s shelve above the micro-centrifuge) by vortexing to
resuspend the resin (not the white band). Slightly open and snap off the bottom of
the column (snap HARD at score)
13. Place tube in open screw cap tube and spin 3K, 1' to clear column. Dispose of tube,
move column to new tube.
14. Add the probe to the top of the column. Spin 3K, 2'. Green color should stay behind.
15. Dispose of column, put screw cap on the tube.
16. Remove 1µL of the probe and place pipette tip in small scintialltion vial. You do not
need to add scintillation fluid. Count in the scint. counter using the user 10 card and
program. Do "count single rack." You expect 200000-400000 counts for that 1 µL.
17. BOIL THE PROBE 5' at 95-100 °C, cool on ice 3-5'.
CHURCH-GILBERT BLOTTING
1. will need to prehyb the blot in 15mL hybridization solution at 65°C in rotator over for
90min. Bottles for hybridization are on sink nearest tc room, prepare by washing
several X in ddH20. These are stored in count off.
2. Add 2x1,000,000 cpm/mL hyb solution in 10mL that is 2x10,000,000. BOIL THE
PROBE BEFORE ADDING TO THE HYB MIX.
3. Hybridize at 65°C O/N
4. Dispose of probe in radioactive waste
5. Rinse blot briefly with 50mL wash solution heated to 65° and dump down sink
(record!)
6. Wash 3X 30' at 65° in rotator oven.
7. Wrap blot in saran wrap and expose to PI screen (2hrs - O/N) or film with screen at 80°C for O/N - several days.
( ALTERNATIVE - QUIK HYB BLOTTING)
1. Add 100µL Stratagene salmon sperm DNA to probe before boiling. Boil 4-5', place on
ice
2. Prehyb in 3mL Quik Hyb solution (Strategene - in Yi's fridge) > 15' at 65°C
3. Add probe to hyb in the 3mL (still 2000000 counts/mL). Hyb 90', 65°
4. wash twice 0.1% SDS, 2X SSC 15' RT
5. wash once 0.1X SSC. ).1% SDS 55-60°, 15' (To heat solution, nuke 1')
STRIPPING
1. Heat 100-150mL 1% SDS in TE to boiling in microwave
2. Add to blot and shake at RT for 30'
3. Discard wash and monitor radioactivity.
4. Repeat until no signal detected
5. Rinse with 2X SSC and prehyb again.
RECIPES
Loading Buffer:
50µL formamide
18µL formaldehyde
12µL H20
10µL 10X MOPS
DEPC water:
DEPC is in the cold box, left side near the bottom
Add 1:1000 to fresh ddH2O from the spigot in 1L bottles (good to make about 2L to
start)
Keep at 37°C in warm room for several hours to O/N, then autoclave.
10X MOPS DEPC
Dissolve 6.8g anhydrous NaOAc in 800mL H20
pH to 7.0 with 10N NaOH
add 20mL 0.5M EDTA pH 8.0
DEPC treat 37°C O/N (1:1000)
autoclave
add 41.93g MOPS
recheck pH (7.0)
fill to 1L with DEPC H20
filter sterile 0.22µm filter.
cover in foil, store dark. Use only until straw colored
20X SSC:
in 400 mL ddH2O:
87.65 g NaCl
44.1g solium citrate (or 50.25g Na citrate 2H2O)
adjust pH to 7.0 with a few citric acid crystals.
adjust V to 500mL
Methylene Blue
0.04% in 0.5M NaOAc pH 5.3
Acetic Acid
glacial acetic acid is 17.4N, dilute with ddH20 to 1N
Church-Gilbert Hybridization solution:
for 50mL:
7.4mL ddH2O
25mL 1M NaHPO4 pH 7.2
17.5 mL 20% SDS
0.1mL 0.5M EDTA pH 8.0
0.5g BSA (Sigma 7906)
Church-Gilbert Wash Solution:
for 500mL:
450mL ddH2O
20mL 1M NaHPO4 pH 7.2
25mL 20% SDS
1mL 0.5M EDTA pH 8.0