Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit

mMESSAGE mMACHINE® Kit Procedure Capped

transcription reaction assembly1.Thaw the frozen reagentsPlace the RNA Polymerase Enzyme Mix on ice, it is stored in glycerol and will not

be frozen at –20°C.

Vortex the 10XReaction Buffer and the 2XNTP/CAP until they are completely in solution. Once thawed, store the ribonucleotides (2XNTP/CAP) on ice, but keep the 10XReaction Buffer at room temperature while assembling the reaction.

All reagents should be microfuged briefly before opening to prevent loss and/or contamination of material that may be present around the rim of the tube.

2.Assemble transcription reaction at room temp

The spermidine in the 10XReaction Buffer can coprecipitate the template DNA if the reaction is assembled on ice.

Add the 10XReaction Buffer after the water and the ribonucleotides are already in the tube.

The following amounts are for a single 20µL reaction. Reactions may be scaled up or down if desired.

IMPORTANT!The following reaction setup is recommended when the RNA

produced will be 300bases to 5kb in length. For transcripts longer or shorter than this, consider the suggestions in sections“Optimizing yield of long transcripts” 3.Mix thoroughly

Gently flick the tube or pipette the mixture up and down gently, and then

microfuge tube briefly to collect the reaction mixture at the bottom of the tube.

4.Incubate at 37°C, 1hr

Typically, 80% yield is achieved after a 1hr incubation. For maximum yield, we recommend a 2hr incubation. Since SP6 reactions are somewhat slower than T3 and T7 reactions, they especially may benefit from the second hour of incubation.

A second hour of incubation is recommended for synthesis of <300base

transcripts and for inefficiently transcribed templates. (See sections“Optimizing yield of long transcripts” on page 20 and“Optimizing yield of short transcripts” on page 21 for optimizing yield from templates coding RNA outside the 0.3–5kb range.

mMESSAGE mMACHINE® Kit User Guide11