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210μg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 |
10× Reaction Buffer* |
100μL |
-20℃ |
100mM ATP Solution |
100μL |
-20℃ |
100mM GTP Solution |
100μL |
-20℃ |
100mM UTP Solution |
100μL |
-20℃ |
100mM CTP Solution |
100μL |
-20℃ |
Enzyme Mix |
100μL |
-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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操作要点:
注:10× 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* |
10× Reaction Buffer |
2μL |
Enzynme Mix |
2μL |
*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.
参考文献:
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.