| Modified Eberwine ("antisense") RNA Amplification Protocol | | 点击： 作者：51protocol收集 来源： 时间： 2007-03-13 本站论坛 |
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hexamers per labeling reaction.
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10X Second Strand Buffer:
200 mM Tris pH 6.9
900 mM KCl
46 mM MgCl2
1.5 mM Nicotine Adenine Dinucleotide (Calbiochem #481915)
100 mM (NH4)2SO4
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Linear Acrylamide Recipe
(from Joe Derisi, Brown lab)
for 375 uL of 10mg/ml stock:
75 uL 5% acrylamide:
3.75 mg acrylamide
1.5 uL 2M Tris pH 8
0.5 uL 3M NaAc
0.1 uL 0.5M EDTA
73 uL water
add 1 uL 10% ammonium persulfate and 0.1 uL TEMED
polymerize 30 minutes
add 2.5 volumes absolute etoh
spin 5 minutes top speed
aspirate supernatant
redissolve in 375 uL (DEPC) water for final concentration of 10 ug/uL
dilute 1:100 in DEPC water for use in Eberwine antisense RNA amplification
protocol
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References:
Luo, et al., Nature Medicine 5(1):117, 1999.
Carninci, et al., PNAS 95:520, 1998.
Eberwine, et al., PNAS 89:3010, 1992.
Van Gelder, et al., PNAS 87:1663, 1990.
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