CloningofsmallRNAswith5’phosphateand3’OHends3
Load your precipitated PCR samples into 2 consecutive lanes so as not to overload the lanes. For each different sample, I would run a separate ladder so that the gels can be cut and stained separately OR run individual gels for individual samples to avoid contamination.Stain the gels in 0.5X TE /EtBr in a clean container for 4 to 5 minutes.Cut out ~92 bp band with a clean razor blade and put band into a 0.5 ml microt......閱讀全文
Cloning－of－small－RNAs－with－5’－phosphate－and－3’－OH－ends2
3’ Adaptor Ligation and PurificationHeat shock the RNA by putting at 90°C for 30 seconds. Snap cool on ice.Set up the 3’Adaptor ligation reaction in a
Cloning－of－small－RNAs－with－5’－phosphate－and－3’－OH－ends3
Load your precipitated PCR samples into 2 consecutive lanes so as not to overload the lanes. For each different sample, I would run a separate ladder
Cloning－of－small－RNAs－with－5’－phosphate－and－3’－OH－ends01
IntroductionThe following protocol describes a procedure for the purification and cloning of miRNAs and other small RNAs in the? 20-30 nucleotide size
RNAi制備小分子干擾RNA（small－interfering－RNAs，siRNAs)的方法2
越來越多的研究人員開始采用小分子干擾RNA（small interfering RNAs，siRNAs)來抑制特定的哺乳動物基因表達。siRNA是一種短片斷雙鏈RNA分子，能夠以同源互補序列的mRNA為靶目標降解特定的 mRNA，這個過程就是RNA干擾途徑（RNA interference
RNAi制備小分子干擾RNA（small－interfering－RNAs，siRNAs)的方法1
最適用于：快速而經濟地研究某個基因功能缺失的表型不適用于：長時間的研究項目，或者是需要一個特定的siRNA進行研究，特別是基因治療體內表達前面的3種方法主要都是體外制備siRNAs，并且需要專門的RNA轉染試劑將siRNAs轉到細胞內。而采用siRNA表達載體和基于PCR的表達框架則屬于：從轉染到細
RNAi術語表
RNAi GlossaryDicer?- Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs. Dicer processes long dsRNA
Phosphate－Assay
1. Make standards using sodium phosphate at the following uM concentrations: 0, 2, 5, 7, 10, 20, 40, 60, and 80. Use the screw top glass tubes.2. Dry
RNAi相關的名詞解釋[英文]
Argonaute?- A family of proteins containing multiple domains and involved in RNA interference (RNAi). Argonatue is the main component of RNAi effector
Fusion－and－Cloning
Author:?Nanci DonackiSource:?Contributed by Nanci DonackiAbstract:?Procedure for establishing hybridoma in one stepReagents(StemCell Technologies, Inc
Fusion－and－Cloning
ReagentsMedium A - Pre-fusion Medium and Hybridoma Expansion MediumMedium B - Fusion Medium Medium C - Hybridoma Recovery MediumMedium D - Hybridoma S
DNA轉化實驗指導2
1B.??Cloning?1.?????A caveat on dephosphorylation: the most common reason for failure to obtain colonies is a result of adding too much BAP or CIP to
Cloning－PCR－products－using－TA－vectors
Cloning PCR products using TA vectorsby Paul N. Hengen, Ph.D.?*Methods and reagents is a unique monthly column that highlights current discussions in
General－Cloning－Protocols
Large Scale Preps:?(See Large scale plsasmid prep protocol for more details)Cultures: Inoculate a 5 mL LB/Amp (50 - 100 μg/mL) culture in early a.m. w
PCR基本實驗方法（五）
Cloning?PCR ProductsT-A Cloning Strategy:?Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated ad
PCR基本實驗方法（五）
Cloning?PCR?ProductsT-A Cloning Strategy:?Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated ad
Simplified－Calcium－Phosphate－Coprecipitation
Materials2x HEPES: 8 g NaCl, 0.105g NaHPO4, 6.5 g HEPES, H2O to 500 mL, pH 6.95 to 7.10 (try a range)2M CaCl2: store in aliquots at -20°CDNA 4 mg/mLPr
Phosphate－(Sodium)－buffer－Chart
Phosphate (Sodium) buffer ChartStock solution A2 M monobasic sodium phosphate, monohydrate (276g/L)Stock solution B2 M dibasic sodium phosphate (284 g
Phosphate－Assay－by－Suprya－Jaydev
ReagentsAshing buffer:10 g Mg(NO3)2?100 ml EtOH1.5 N HCl stock:119.7 ml concentrated HCl (11.6M @ 36% by weight)880.3 ml H2O1 N Sulfuric acid stock:28
Cloning－by－Limiting－Dilution－of－Hybridoma
Author:?Nanci DonackiSource:?Contributed by Nanci DonackiDate Added:?Tue May 14 2002Date Modified:?Tue Apr 27 2004MaterialsDMEM, high glucose (Life Te
基因cloning經驗指南
我們克隆基因的時候，往往可以通過一些途徑（如pcr,EST庫或者文庫篩選）得到基因的部分片段。然后通過3'race 和 5'race 方法往兩端延伸。有時會出現無法延伸的狀況，如果你超作沒有失誤的話，這時候很有可能是你模板GC含量過高的緣故，普通pcr 是沒有辦法延伸的，可以用擴高g
Genomic－Cloning－Technical－Manual
Genomic Cloning Technical ManualAn optimal strategy for genomic cloning should meet three requirements: 1) a maximum number of recombinants should be
Oxidative－reactions－of－the－pentose－phosphate－pathway
One form of chemical energy used to drive biosynthetic reactions forward is the reducing power of the energy carrier NADPH. NADPH is essential to driv
NAi－protocol
siRNA protocolsOur current strategy with siRNA is to synthesis relatively small amounts enzymatically and use these to test for efficiency by western
RNAi－protocol
?siRNA protocolsOur current strategy with siRNA is to synthesis relatively small amounts enzymatically and use these to test for efficiency by western
鵪鶉PIWI基因的克隆及其結合小RNA的鑒定
PIWI 蛋白能通過與多種內源非編碼小RNA結合在表觀水平和轉錄后水平調控基因的表達。然而在家禽中，PIWI蛋白的生物學功能及其結合小RNA的作用機制尚不清楚。揚州大學動物科學與技術學院陳國宏教授課題組對該作用機理進行了研究*，研究成果發表在2012年12月刊的PLoS ONE上。PIWI 基因
“電子”基因克隆－（sillcon－cloning）
利用計算機來協助克隆 基因，稱為“電子”基因克隆 （sillcon cloning），是與定位克隆 、定位候選克隆 策略并列的方法之一，即采用生物信息學的方法延伸EST序列，以獲得基因部分乃至全長的cDNA序列。EST數據庫的迅速擴張，已經并將繼續導致識別與克隆 新基因策略發生革命性變化。1
磷酸緩沖液（phosphate－buffer）
按照下表所給定的體積，混合1 mol/L 的磷酸二氫鈉（單堿）和1mol/L 磷酸氫二鈉（雙堿）貯液，獲得所需pH的磷酸緩沖液。配制1 mol/L 的磷酸二氫鈉（NaH2PO4?H2O）貯液：溶解138g于足量水中，使終體積為1L;1mol/L 磷酸氫二鈉（Na2HPO4）貯液:溶解14
Experimental－Protocol－for－cDNA－Library－Construction
Experimental Protocol for cDNA Library ConstructionIdentify appropriate celltype over-expressing corresponding gene.Find out if transcription can be s
非編碼small－RNA參與缺氧下的血管生成（一）
非編碼RNA是近年來轉錄組學研究的熱點，其中，long non-coding RNA（lncRNA），microRNA，circularRNA是大家研究的非常多的非編碼。其實，在small non-coding RNAs世界，除了我們熟知的microRNA之外，還包括piwi-interac
Detection－of－apoptotic－process－in－situ－using－immunocytochemical
1. INTRODUCTION??Apoptosis was observed from invertebrates to lower and higher verterbrates, and intervenes both in physiological and in pathological