Technical Bulletin: #156
|In Vitro Synthesis of RNA |
SP6, T7 and T3 phage RNA polymerases have high specificity for their respective 23 base promoters (Figure 1). The development of cloning vectors containing promoters for these polymerases has made the in vitro synthesis of single stranded RNA molecules a routine laboratory procedure. Modern multipurpose cloning vectors contain a multiple cloning site (MCS) flanked on each side by promoters for different polymerases. This allows the synthesis of either sense or antisense RNAs from sequences cloned into the multiple cloning site. Plasmid templates are generally linearized with a restriction enzyme to allow run off RNA transcripts to be synthesized with a defined end. In addition to plasmid DNA, PCR products and synthetic oligonucleotides can be used as templates for transcription reactions. For PCR products, one of the primers needs to include the promoter sequence of a phage polymerase so that the PCR product contains a phage promoter. Synthetic oligonucleotides need to contain the phage promoter, which must be double stranded. However, the remainder of the template need only be single stranded.
RNA Probe Synthesis with MAXIscript and MEGAscript Kits
Synthesis of Large Mass Amounts of RNA - A Complete Selection of Kits
mMESSAGE mMACHINE™ Kits have been specifically designed for high yield synthesis of capped RNA for use in microinjection and in vitro translation studies. They utilize the same high yield technology as the MEGAscript Kits, and contain cap analog premixed with nucleotides in a single tube (4:1 Cap:GTP). The mMESSAGE mMACHINE Kit provides a simplified format for the routine synthesis of 15 to 35 µg of capped RNA per 20 µl reaction. Use the MEGAscript Kit when you need to manipulate the cap analog:GTP ratio. Cap analog is available separately for use in these applications.
Capped vs. Uncapped RNA Transcripts
The MEGAshortscript™ Kit is designed specifically to give high yields of small RNAs from short templates (< 300 bases). Synthesis from a given mass amount of RNA from a small template requires more initiation events than for a larger template. Since initiation is generally the rate-limiting step in an in vitro transcription reaction, the yields of small RNAs are normally relatively low. The MEGAshortscript Kit utilizes a high polymerase enzyme mix to maximize yields of small transcripts. MEGAshortscript Kits work well with oligonucleotide or PCR product templates as well as standard plasmid templates.