Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit Introduction

Materials provided

with the kit

Materials not

provided with the

kit

6The mMESSAGE mMACHINE® Kit should be stored in a non-frost-free freezer. Keep all reagents on ice while using the kit; the nucleotides and enzymes are especially labile. Reagents are included for 25 mMESSAGE mMACHINE® reactions.AmountComponentStorage1.75mLNuclease-free Waterany temp 50µLEnzyme Mix (SP6, T7, or T3) :–20°CBuffered 50% glycerol containing RNA polymerase, RNase Inhibitor, and other components50µL10X Reaction Buffer (SP6, T7, or T3) –20°Csalts, buffer, dithiothreitol, and other ingredients250µL2X NTP/CAP (SP6, T7, or T3) : a neutralized buffered solution –20°Ccontaining:SP6 KitsT7 and T3 KitsATP10mM15mMCTP10mM15mMUTP10mM15mMGTP2mM3mMcap analog8mM12mM100µLGTP 20mM in SP6 Kits; 30mM in T3 and T7 Kits–20°C100µLTURBO DNase (2U/µL)–20°C10µLpTRI-Xef, 0.5mg/mL (Control Template)–20°C1mLAmmonium Acetate Stop Solution–20°C5M ammonium acetate, 100mM EDTA1.4mLLithium Chloride Precipitation Solution–20°C7.5M lithium chloride, 50mM EDTA1.4mLGel Loading Buffer II: a 1–2X gel loading solution for TBE –20°Cpolyacrylamide and agarose gels 95%formamide0.025%xylene cyanol, 0.025% bromophenol blue18 mM EDTA0.025% SDS Components are specifically calibrated for each lot and kit type. Mixing components from different lots, or from kits for different enzymes (SP6, T7, T3) will compromise RNA yield. Store Nuclease-free Water at –20°C, 4°C or room temp. DNA template: The DNA template must have the correct RNA polymerase promoter site (T7, T3, or SP6) upstream of the sequence to be transcribed. The suggested template concentration is 0.5µg/µL in water or TE (10mMTris-HCl (pH7–8), 1mMEDTA).mMESSAGE mMACHINE® Kit User Guide