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1) Speed vac 30 ug total RNA
2) Resuspend in 30 ul hybridization buffer and probe.
To make 1 ml
80% deionized formamide 800 ul formamid-e
0.4 M NaCl 100 ul 4M NaCl
1 mM EDTA 2 ul .5M EDTA
40 MM PIPES pH 6.7 40 ul im PIPES
+ Probe 58 ul H20 + Probe
1000 ul
3) Add enough probe to give 50, 000 cpm/sample - initial tests should be performed to determine that reaction is in probe excess.
4) Heat to 850C x 5 minutes
" Turn H20 bath down to 500C H20 bath and incubate for 12-16 hours.
6) RNAase A type IIIA (Sigma) make 2 mg/ml
stock - boil x 10 min - store at 40C.
7) Add RNAase-solution - 350 ul per sample
To make
Working Stock 10 ml
RNAase A 40mg/ml 2 mg/ml 200 ul
Tris pH 7.5 iomm im 100 ul
EDTA imm .5m 2 ul
NaCl 0.2M 5M 400 ul
LiCl2 O.lml 5m 200 ul
H20 9.1 ml
10 ml
8) Vortex, cfg
9) Place in 250C H20 x 30 minutes
10) Add 10 ul 20% SDS
+ 10 ul Prot K (10mg/ml)
11) Heat to 370C for 10-15 minutes
12) P: CIAA lx
13) Add 10 ug TRNA (i ul of 10 ug/ul stock)
2 volumes ETOH
-700C x 30 minutes
-Spin at 40C x 5 min
-Wash with 500 ul 70% ice cold ETOH -Spin at 40C -Dessicate (speed vac)
14) Resuspend in 5 ul loading buffer
850 ul formamide
50 ul xylene cyanol 10%
50 ul bromophenol blue 1%
100 ul TBE (10x)
1050 ul
15) Heat sample to 900C x 5 minutes
16) Transfer to ice
17) Load 3 ul on 6% PAGE
18) Run at 1500 V 4 hours
19) Separate plates, lift gel on filter paper, wrap in saran wrap
20) Dry under vaccum at 800C x 30 minutest hour
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