AmpliCap-Max™ T7 High Yield Message Maker Kit

Store at -20°C in a freezer without a defrost cycle. Do not store at -70°C

The AmpliCap-Max™ T7 High Yield Message Maker Kit produces capped RNA by in vitro transcriptionusing T7 RNA polymerase and the standard cap analog m7G[5']ppp[5']G. A 30 minute, 20 μl AmpliCapMax reaction yields up to 60 μg of m7G-capped RNA from 1 μg of the 1.4 kb standard T7 control templateDNA. The m7G[5']ppp[5']G cap analog/NTP PreMix contains all four ribonucleotides and the monomethylatedcap analog. The PreMix ensures the optimal concentration of each NTP and ratio of methylated capanalog to GTP (4:1), maximizing transcript capping (~80%) and yield. Because the concentration of GTPin the reaction is limiting, the cap analog is preferentially incorporated as the first or 5'-terminal G of thetranscript. CELLSCRIPT also offers the MessageMAX™ T7 ARCA-Capped Message Transcription Kit which alsoproduces up to 60 μg of capped RNA in a 30 minute standard control reaction with an 80% cappingefficiency. The MessageMAX Kit uses the anti-reverse cap analog (ARCA), m27,3'-OG[5']ppp[5'].

RNase-Free DNase I is provided in a 50% glycerol solution containing 50 mM Tris-HCl, pH 7.5,10 mM CaCl2, 10 mM MgCl2 and 0.1% Triton® X-100. ScriptGuard RNase Inhibitor is supplied in a50% glycerol solution containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM DTT, 0.1 mM EDTAand 0.1% Triton® X-100. All other enzymes are provided in a 50% glycerol solution containing 50 mMTris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100.

Unit Definitions One unit of RNase-Free DNase I digests 1 µg of pUC19 DNA to oligodeoxynucleotides in 10 minutesat 37°C. One unit of ScriptGuard RNase Inhibitor results in 50% inhibition of 5 ng of RNase A. Activity ismeasured by the inhibition of hydrolysis of cyclic 2',3'-CMP by RNase A. Functional Testing The AmpliCap-Max T7 High Yield Message Maker Kit is functionally tested under standard reactionconditions using the T7 Control Template DNA. The kit must produce at least 40 µg of RNA from1 µg of the T7 Control Template DNA in 30 minutes at 37°C. Contaminating Activity Assays All components of the AmpliCap-Max T7 High Yield Message Maker Kit are free of detectable RNaseand DNase activity, except for the inherent activity of the RNase-Free DNase I component.