果蝇RNAi的实验中双链短RNA的合成（dsRNA)方法.pdfVIP

中国试剂网 0 果蝇RNAi 的实验中双链短RNA 的合成 （dsRNA)方法 本文来自于哈佛大学医学院果蝇RNAi 筛选中心的经典实验方法，专门用于果蝇 RNAi 实验方法。感谢哈佛大学医学院果蝇RNAi 筛选中心的支持！ PrimerDesigneddsRNA TemplateSelection PCR InvitroRNATranscription dsRNAPurification,Quantification,Storage dsRNAFromClones PCR InvitroRNATranscription Purification,Quantification,Storage AdditionalReferences We routinely produce dsRNA by in vitro transcription of a PCR generated DNA template containing the T7 promoter sequence on both ends (I. Primer Designed dsRNA).ItisalsopossibletoproducedsRNAusingPCRgeneratedDNAtemplates containingeithertheT7SP6ortheT7T3promotersoneitherend(II.dsRNA FomClones).Thismethodislessefficient,especiallywhenworkingonalargescale. **Allworkshouldbedoneinasterile,RNasefreeenvironment,usingonlysterile, RNase free solutions and materials, and while wearing gloves do reduce contamination. I.PrimerDesigneddsRNA A.TemplateSelection TemplatescanbegeneratedbyPCRoncDNA(includingESTsfromBDGP),genomic DNA,orfirststrandRTcDNA.MostofthedsRNAshouldcorrespondtoexonsbut dsRNA with two or more exons interrupted by introns will also work well. We generally aim for ~500 bp products although RNAi with products ranging from 1503000bp have been shown to work. The target sequence should not contain complete21merhomologytoothergenesoryourdsRNAcouldbenonspecific.One 中国试剂网 0 casehasbeenseenwherethetemplatecontainedonemismatchina21meridentical stretch to another gene, and still only the level of the intended target was reduced, while the close homologue was unaffected. Since the targeted region within a transcriptmayinfluencethesuccessofRNAi,thesafest approachistousedsRNA correspondingtothe5or3UTR.Itisrecommendedtocheckthevarioussequence sources(Publications,NCBIandBDGP,genomicandESTs)toconfirmtheprimary sequenceofagivengene. We