| Protocol for mRNA amplification，非常详细的RT-PCR实验过程[Howard Hughes Medical | 点击： 作者：51protocol收集 来源： 时间： 2007-03-19 本站论坛
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Add 128uL to each tube.
37C for 5min to digest mRNA, 94C for 2min to denature, 65C for 1min for specific priming and
75C for 30min for extension.
Stop reaction with 7.5ul 1M NaOH.
Incubate at 65C for 10min to inactivate enzyme.
DS cDNA cleanup:
Add to PCR Tube
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Ammt |
Reagent |
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1ul |
0.1ug/ul Linear Acrylamide (Ambion Cat# 9520) |
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150ul |
Phenol: Chloroform: Isoamyl alcohol 25:24:1 (Boehringer Mannheim Cat #101001) |
Mix well by pipetting (be careful not to spill or contaminate).
Spin the Phase lock gel tube down for 30 seconds at maximum speed.
Transfer the slurry solution to Phase lock gel tube (5’-3’ Inc. Cat# p1-257178)
Spin at 14,000rpm for 5min at room temperature.
Transfer the aqueous phase to RNase/DNase-free tube (stopping point).
Add 70ul of 7.5M ammonium acetate (Sigma Cat# A2706) and gently mix.
Add 1ml 95% room temperature ethanol.
Centrifuge at 14,000rpm for 20min at room temperature.
Wash pellet with 500ul 95% ethanol and spin pellet down at maximum speed for 6min.
Air dry pellet and resuspend ds cDNA in 8ul DEPC H2O (stopping point).
In Vitro TRanscription
(Ambion; T7 Megascript Kit #1334)
In PCR reaction tube, mix
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