RACE操作步骤.doc

Storage ConditionsStore Control Human Placental Total RNA and SMARTer II A Oligonucleotide at 70C. Store the NucleoTrap Gel Extraction Kit at room temperature.Store all other reagents at 20C. IMPORTANT: Prior to cDNA synthesis, please make sure that your RNA is intact完好的 and free of contaminants (see Section V.C. Assessing the Quality of the RNA Template).Do not change the size (volume) of any of the reactions. All components have been optimized for the volumes specified.1.10l体系，PCR管冰上配制，混匀并瞬时离心，室温放至第7步 2.0 l 5X First-Strand Buffer 1.0 l DTT (20 mM) 1.0 l dNTP Mix (10 mM) 4.0 l 总体积2. 新PCR管分别混匀以下试剂5-RACE-Ready cDNA 3-RACE-Ready cDNA1.02.75 l RNA* 1.03.75 l RNA*1.0 l 5-CDS Primer A 1.0 l 3-CDS Primer A3.加无菌水至3.75 l 加无菌水至4.75 l 4.混匀并瞬时离心 混匀并瞬时离心 5 PCR仪：72 3min，42 2min，瞬时离心14000g，10s（12000rpm，10s）. 6. 5-RACE-Ready cDNA 每反应加1.0ul SMARTer A oligo 7. 4.0 l Buffer Mix from Step 1 0.25 l RNase Inhibitor (40 U/l) 1.0 l SMARTScribe Reverse Transcriptase (100 U) 5.25 l 总体积8. 5-RACE 3-RACE5.25ul Master Mix from step 5.25ul Master Mix from step4.75l 5 RACE cDNA from step 4.75ul3-RACE cDNA from step10l总体积 10l总体积9. 轻轻吸打混匀，并瞬时离心10.PCR仪：42 90min 11. PCR仪：70C 10 min. 12. 稀释反应产物：用Tricine-EDTA buffer如果起初的总RNA200ng，则加20l进行稀释如果起初的总RNA200ng，则加100l进行稀释（本试验采用的100l稀释）如果起初用的是PolyA+ RNA，则加250l进行稀释13.稀释后的样品可以放-20保存（保质期3个月）VI. Rapid Amplification of cDNA Ends (RACE)cDNA末端快速扩增（RACE）1.PCR管，50l体系，冰上混匀以下试剂：34.5 l PCR-Grade Water5.0 l 10X Advantage 2 PCR Buffer 1.0 l dNTP Mix (10 mM; in SMARTer RACE or Advantage 2 PCR Kit) 1.0 l 50X Advantage 2 Polymerase Mix41.5 l 总体积2. 枪头轻轻吸打混匀，无气泡，瞬时离心3. 5-RACE PCR体系：5 -RACE-Ready cDNA2.5ulUPM(10*)5.0ul5 RACE引物（10M）1.0ulMaster Mix 41.5ul总体积50.0ul吸打混匀，瞬时离心3-RACE-Ready cDNA2.5ulUPM(10*)5.0ul3RACE引物（10M）1.0ulMaster Mix 41.5ul总体积50.0ul吸打混匀，瞬时离心4. PCR反应（热启动）：94 5min94 30s61 40s自己设计的引物的温度72 3min 2 40cycle72 10min4 10h PCR产物可以-20保存第二轮PCR或巢式PCR1.将第一轮PCR产物用Tricine-EDTA稀释50倍(吸取第一轮PCR产物5.0l，加到245l的Tricine-EDTA Buffer中，将剩余的-20保存)2. 混匀以下试剂PCR-Grade Water36ul10X Advantage 2 PCR Buffer5.0uldNTP Mix （10mM）1.0ul50X Advantage 2 Polymerase Mix（5U/ul）1.0ul稀释50倍的PCR产物5.0ulNUP Primer（10uM）1.0ul5或3RACE引物(10uM）1.0ul总体积50ul枪头吸打混匀，瞬时离心。3.PCR反应（热启动）：94 5min94 30s57 40s72 3mi