SolexaSmallRNA樣品制备标准操作规程.doc

Solexa Small RNA 样品制备标准操作规程（第3版） Small RNA样品制备组 摘要：Solexa Small RNA样品制备主要包括以下几个内容：1）将总RNA跑TBU变性胶，回收16－30nt的Small RNA；2）5 adaptor 连接并纯化回收连接产物；3） 3 adaptor 连接并纯化回收连接产物；4）RT－PCR并回收～92bp大小的条带。整个流程需要约4天时间。 1 试剂及耗材 1.1 试剂 1.1.1购买试剂及耗材 厂家 货号 存储条件 SuperScript? II Reverse Transcription Kit (10,000 Units) Invitrogen 18064-014 -20℃ RNase OUT? (5000U) Invitrogen 10777-019 -20℃ 14-30ssRNA Takara D3416 -80℃ 25 bp DNA ladder (50 μg) Invitrogen 10597-011 -20℃ 10bp DNA ladder (50 μg) Invitrogen 10821-015 -20℃ Novex? 15% TBE-Urea Gel, 1.0 mm/10 well Invitrogen EC6885BOX 4℃ Novex? 10% TBE-Urea Gel, 1.0 mm/10 well Invitrogen EC6875BOX 4℃ 6% Novex? TBE PAGE Gel, 1.0 mm, 10 well Invitrogen EC6265BOX 4℃ NEBuffer 2 stock(10x) NEB B7002S -20℃ DEPC-H2O Ambion AM9915G 4℃/RT TE pH 8.0 Buffer (500 ml) Ambion AM9849 4℃/RT Pellet Paint? NF Co-precipitant Novagen 70748-4 -20℃ Spin-X Cellulose Acetate Filter (2 ml, 0.45 μm) Fisher 07-200-388 RT 10 mM dNTP mix NEB N0447L -20℃ DEPC Calbiochem 298711 4℃ T4 RNA Ligase1 NEB M0204S -20℃ T4 RNA Ligase (500U, 10U/ul) Promega M1051 -20℃ T4 RNA Ligase (200 U，30U/ul) Takara D2050 -20℃ 2x Gel Loading Buffer II Ambion AM8546G -20℃ 5 M NaCl (100 mL) Ambion AM9760G 4℃/RT Glycogen (5 x 1 mL) Ambion AM9510 -20℃/-80℃ 2xPhusion HF master mix Finnzymes F-531S -20℃/-80℃ 5’ RNA adaptor (25 μM) Takara 合成 -80℃ 3’ RNA adaptor (25 μM) Takara 合成 -80℃ Small RNA RT-primer (?100 μM) Takara 合成 -20℃ Small RNA P5 long primer(100 μM) Takara 合成 -20℃ Small RNA P7 primer (100 μM) Takara 合成 -20℃ 无水乙醇 国产 RT 1000ul Sterile, RNAase-free pipette tips Axygen RT 200ul Sterile, RNAase-free pipette tips Axygen RT 10ul Sterile, RNAase-free pipette tips Axygen RT 2ml Sterile, RNAase-free microtubes Axygen MCT-200-C RT 1.5ml Sterile, RNAase-free microtubes Axygen MCT-150-C RT 0.2ml Sterile, PCR microtubes Axygen PCR-02D-C RT 0.5ml non-stick Sterile, RNAase-free microtubes Ambion AM12340 RT VWR 加样枪头 VWR ? RT RNA zap Ambion AM9890 RT Pure EtBr(10mg/ml) I