Life Ambion T7启动子转录试剂盒说明书

AAppendix A Supplemental InformationAdditional procedures

Optimizing yield of

long transcripts

20If the transcription reaction contained a radiolabeled nucleotide tracer (e.g. [α-32P]UTP), the RNA can be visualized by autoradiography. Agarose gels should be dried before exposing to X-ray film, but thin (0.75mm thick) polyacrylamide gels may be transferred to filter paper, covered with plastic wrap, and exposed directly (when 32P is used). Approximate exposure times for visualizing low specific activity transcripts (e.g. when 1µL of 800Ci/mmol, 10mCi/mL [α-32P] UTP was used in the mMESSAGE mMACHINE reaction) are about 10–30min with an intensifying screen, or several hours to overnight without a screen, when 1 µL of the undiluted reaction is run on the gel. A recipe for standard denaturing (i.e. 8M urea) polyacrylamide gels is given in section“Denaturing acrylamide gel mix” on page 25.When synthesizing transcripts that are larger than 5or6kb, GTP will become rate limiting and may result in low yield, premature termination of transcription, or both. To circumvent this, it may be desirable to supplement the reaction with extra GTP. Shown below is the effect of adding the indicated volumes of the GTP supplied with the kit to an otherwise ordinary mMESSAGE mMACHINE reaction.Added GTP*Final Cap Fraction of Analog:GTP RatioTranscripts Capped0µL4.0:180%1µL2.0:167%2µL1.3:157%3µL1.0:150%*The GTP supplied with T7 and T3 mMESSAGE mMACHINE Kits is 30mM. The GTP supplied with SP6 mMESSAGE mMACHINE Kits is 20mM1.How much additional GTP should be added?For templates in the 5–8kb range we suggest initially testing the addition of 1µL of GTP. For larger templates you should try titrating additional GTP to determine the minimum amount needed. Adding GTP will decrease the fraction of transcripts synthesized with a cap, but it will result in higher yields of full length product. The fraction of capped transcripts is proportional to the ratio of Cap Analog to GTP in the transcription reaction.2.There is a trade-off between RNA yield, and good capping efficiencyWe tested the effect of varying the ratio of cap analog to GTP on the yield of RNA from transcription reactions and on the efficiency of translation of the resulting RNA in a reticulocyte lysate in vitro translation system (See Table1). The translation of globin RNA in a reticulocyte lysate is known to be very cap dependent. As the ratio of cap analog to GTP increases, the yield of RNA decreases. Conversely, the translational efficiency of the RNA synthesized increases with increasing cap analog to GTP ratios. This reflects the increasing fraction of transcripts with a 5' cap. Note that a cap analog to GTP ratio of 4:1 provides a good compromise between RNA yield, and capping efficiency in the case of globin. The presence of uncapped transcripts in Xenopus oocyte microinjection experiments does not usually present a problem. These uncapped transcripts are presumably rapidly degraded by the oocyte.mMESSAGE mMACHINE® Kit User Guide