RACE操作步骤.doc
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1、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 (se
2、e 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
3、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 In
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