Northern Protocol
8/20/98
RNA
HARVESTING
(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
FORMALDEHYDE
GEL
1.
Treat (or wash) 250mL flask and a stir bar with DEPC water
2. Treat the medium gel box and
combs with 3% H2O2 in DEPC water for 30', then rinse in DEPCwater
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. 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 soap and fresh ddH2O. Wash gel 6X
fresh ddH2O.
12.
Wash gel 20X SSC for 30'.
13. Assemble turbo blotter (Yi's
bench, paper in cabinet near Fred's bench) according to the manufacturer's
directions. (wash with soapy water and 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 L 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 ddH2O for a final volume of 45µL
3.
Denature 95-100¡C for 2-3 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
Mireya's cabinet) 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.
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