T7体外转录试剂盒 加帽mMESSAGEmMACHINE T7 kit (Ambion(8)

Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit

mMESSAGE mMACHINE® Kit Procedure

mMESSAGE mMACHINE® Kit ProcedurePreparation of template DNALinearized plasmid DNA, and PCR products that contain an RNA polymerase 8promoter site can be used as templates for in vitro transcription with the mMESSAGE mMACHINE® Kit. In general, any DNA with a promoter site, that is pure enough to be easily digested with restriction enzymes can be used for in vitro transcription.Figure 1Phage Polymerase Promoters: Minimal Sequence RequirementsT7+1TAATACGACTCACTATAGGGAGAThe +1 base (in bold) is the first base incorporated into RNA during SP6+1transcription. The underline shows the ATTTAGGTGACACTATAGAAGNGminimum promoter sequence needed for efficient transcription.T3+1AATTAACCCTCACTAAAGGGAGA-17+6Template sizeThe mMESSAGE mMACHINE® Kit is designed to function best with templates that code for RNA transcripts in the 0.3 to 5kb range. The kit can be used to produce shorter, or longer RNA, but modify the reaction as described in section “Optimizing yield of short transcripts” on page 21.OrientationIf sense RNA is needed, it is important to transcribe using the RNA polymerase corresponding to the phage promoter at the 5', or amino-terminal side of the coding region of the protein (using promoter1 in the diagram below). If the template consists of a plasmid, it should be linearized in the polylinker at the opposite (3' or carboxy-terminal side) of the protein-coding region.Antisense (mRNA-complementary) transcripts will be synthesized if the RNA polymerase corresponding to the RNA phage promoter at the 3', or carboxy-terminal side of the coding region of the protein is used (using promoter2 in the diagram below).5'ATG............AAAAAA3'promoter 1promoter 23'5'Transcription using the RNA polymerase corresponding to promoter1 will make sense RNA (the same sequence as the mRNA). If the RNA polymerase for promoter2 is used, antisense RNA will be transcribed.mMESSAGE mMACHINE® Kit User Guide