产品描述:The High Yield T7 RNA Synthesis Kit与传统的体外转录反应相比,有着很高的合成量,能够多达25倍,每个反应可以合成高达180μg的RNA。
试剂盒中提供的原料:包括50个反应,20μL/反应。
Component | 50 rxn kits | Storage |
5× Reaction Buffer* | 200μL | -20℃ |
100mM ATP Solution | 100μL | -20℃ |
100mM GTP Solution | 100μL | -20℃ |
100mM UTP Solution | 100μL | -20℃ |
100mM CTP Solution | 100μL | -20℃ |
Enzyme Mix | 75μL | -20℃ |
Control Template(0.5μg/μL) | 10μL | -20℃ |
DNase I (RNase-free, 1U/μL) | 50μL | -20℃ |
Nuclease-free H2O | 1mL | -20℃ |
Ammonium Acetate Stop Solution | 1.5mL | -20℃ |
Lithium Chloride Precipitation Solution | 1.5mL | -20℃ |
Gel Loading Buffer | 0.5mL | -20℃ |
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注:5× Reaction Buffer储存在-70℃,可能会产生白色沉淀,如果有白色沉淀产生,将试剂管加热至37℃ 5min 并且充分混合使其溶解后使用。
1. 按下表中的量完成20μL反应,可根据需要按照比例放大或者减小
Component | Amount |
Nuclease-free H2O | to 20μL |
100mM ATP Solution | 2μL |
100mM GTP Solution | 2μL |
100mM CTP solution | 2μL |
100mM UTP solution | 2μL |
Template DNA | 1μg* |
5× Reaction Buffer | 4μL |
Enzynme Mix | 1.5μL |
*转录产量取决于模板的添加量。
2. 充分混合,37℃反应 2h。
3.(可选择)加入1μL DNase I(RNase-free),充分混合,37℃反应30min。
储藏温度:-20℃
质控检测:All components are tested in a functional assay as described in this procedure. A 20-μL reaction containing 1 μg of the control template DNA which codes for a ~800b transcript synthesized >150 μg of RNA after a 2 hr incubation.
1. Milligan JF, Groebe DR, Witherell GW, and Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA template. Nucl. Acids Res. 15: 8783–8798.
2. Molecular Cloning, A Laboratory Manual, 2nd edition. (1989) editor C Nolan, Cold Spring Harbor Laboratory Press.