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1、Promega Corporation 2800 Woods Hollow Road Madison, WI 53711-5399USA Telephone608-274-4330 Toll Free800-356-9526 Fax608-277-2516 I PRODUCT USE LIMITATIONS, WARRANTY, DISCLAIMER Promega manufactures products for a number of intended uses. Please refer to the product label for the intended use stateme
2、nts for specific products. Promega products contain chemicals which may be harmful if misused. Due care should be exercised with all Promega products to prevent direct human contact. Each Promega product is shipped with documentation stating specifications and other technical information. Promega pr
3、oducts are warranted to meet or exceed the stated specifications. Promegas sole obligation and the customers sole remedy is limited to replace- ment of products free of charge in the event products fail to perform as warranted. Promega makes no other warranty of any kind whatsoever, and SPECIFICALLY
4、 DISCLAIMS AND EXCLUDES ALL OTHER WAR- RANTIES OF ANY KIND OR NATURE WHATSOEVER, DIRECTLY OR INDIRECTLY, EXPRESS OR IMPLIED, INCLUDING, WITHOUT LIMITATION, AS TO THE SUITABILITY, PRODUCTIVITY, DURABILITY, FITNESS FOR A PARTICULAR PURPOSE OR USE, MER- CHANTABILITY, CONDITION, OR ANY OTHER MAT- TER WI
5、TH RESPECT TO PROMEGA PRODUCTS. In no event shall Promega be liable for claims for any other damages, whether direct, incidental, foresee- able, consequential, or special (including but not lim- ited to loss of use, revenue or profit), whether based upon warranty, contract, tort (including negligenc
6、e) or strict liability arising in connection with the sale or the failure of Promega products to perform in accordance with the stated specifications. Part# 9PIE133 Revised 6/13 Part# 9PIE133 Printed in USA Revised 6/13 pmirGLO Dual-Luciferase miRNA Target Expression Vector: Cat.#Size E133020g Cat.#
7、 E1330 contains: Part No.Name Cat.# E1330 contains: Part No.Name E133ApmirGLO Vector20g C838AOligo Annealing Buffer1ml Description: The pmirGLO Dual-Luciferase miRNA Target Expression Vector(ad)is designed to quantitatively evaluate microRNA (miRNA) activity by the insertion of miRNA target sites 3
8、of the firefly luciferase gene (luc2). These target sites can be introduced by cloning putative miRNA binding sites alone, or the 3 untranslated region (UTR) of a gene of interest, to study the influence of these sites on transcript stability and activity. Firefly luciferase is the primary reporter
9、gene; reduced firefly luciferase expression indicates the binding of endogenous or introduced miRNAs to the cloned miRNA target sequence. This vector is based on Promega dual-luciferase technology, with firefly luciferase (luc2) used as the primary reporter to monitor mRNA regulation and Renillaluci
10、ferase (hRluc-neo) acting as a control reporter for normalization and selection. This vector contains the following features: Human phosphoglycerate kinase (PGK) promoter provides low translational expression, which is advantageous when reduction of signal is the desired response. The PGK promoter i
11、s a nonviral universal promoter, which functions across cell lines (yeast, rat, mouse and human). Firefly luciferase reporter gene (luc2) inversely reports miRNA activity in mammalian cells. Multiple cloning site (MCS) is located 3 of the firefly luciferase reporter gene (luc2). Humanized Renilla lu
12、ciferase-neomycin resistance cassette (hRluc-neo) is used as a control reporter for normalization of gene expression and stable cell line selection. Amprgene allows bacterial selection for vector amplification. SV40 late poly(A) signal sequence is positioned downstream of luc2to provide efficient tr
13、anscription termination and mRNA polyadenylation. Synthetic poly(A) signal/transcription stop site. Concentration: 1g/l in 10mM Tris-HCl, 1mM EDTA; final pH 7.4. GenBankAccession Number: FJ376737. Storage Conditions: See the storage temperature and expiration date on the Product Information Label. Q
14、uality Control Assays Functional Assays Identity Assay: The vector has been sequenced completely and has 100% identity with the published sequence available at: Restriction Digestion: The functional purity of this vector DNA is verified by complete digestion with restriction enzymes at the optimal t
15、emperature for 1 hour. Samples are examined by agarose gel electrophoresis, comparing cut and uncut vector DNA with marker DNA. Contaminant Assays Contaminating Nucleic Acids: RNA, single-stranded DNA and chromosomal DNA are not evident in specified quantities of this vector as determined by agarose
16、 gel electrophoresis. Nuclease Assay: Following incubation of 1g of this vector in Restriction Enzyme Buffer at 37C for 1624 hours, no evidence of nuclease activity is detected by agarose gel electrophoresis. Physical Purity: A260/A2801.80, A260/A2501.05. 20082013 Promega Corporation. All Rights Res
17、erved. Dual-Glo is a registered trademark of Promega Corporation. GeneClip and PureYield are trademarks of Promega Corporation. GenBank is a registered trademark of US Department of Health and Human Services. Products may be covered by pending or issued patents or may have certain limitations. Pleas
18、e visit our Web site for more information. All specifications are subject to change without prior notice. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products. (a)BY USE OF THIS PR
19、ODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence of the luciferase gene except that researchers may (1) create fused gene sequences, and (2) insert and remove nucle
20、ic acid sequences in splicing research. No other use or transfer of this product or derivatives is authorized. Researchers must either (1) use luminescent assay reagents purchased from Promega for all determinations of luminescence activity of this product and its derivatives; or (2) contact Promega
21、 to obtain a license for use of the product. For any uses outside this label license, contact Promega for supply and licensing information. This product is for research use only; no commercial use is allowed. For a full copy of this label license, including the definition of “commercial use,” go to:
22、 (b)U.S. Pat. No. 5,670,356. (c)U.S. Pat. No. 8,008,006 and European Pat. No. 1341808. (d)The method of recombinant expression of Coleopteraluciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673. A license (from Promega for research reagent products and from The Regents of the Un
23、iversity of California for all other fields) is needed for any commercial sale of nucleic acid contained within or derived from this product. Certificate of Analysis Signed by:Signed by: J. Stevens, Quality Assurance Promega Corporation 2800 Woods Hollow RoadMadison, WI 53711-5399 U.S.A. Toll Free i
24、n the USA 800-356-9526 Telephone 608-274-4330 Part# 9PIE133 Printed in USA Revised 6/13 Features List and Map for the pmirGLO Vector SV40 late poly(A) signal106327 SV40 early enhancer/promotor426844 hRluc-neo fusion protein coding region8892664 Synthetic polyadenylation signal27282776 -lactamase (Am
25、pr) coding region 30373897 ColE1-derived plasmid origin of replication40524088 Human phosphoglycerate kinase promoter50945609 luc2reporter gene56457297 Multiple cloning site (MCS, Figure 1)73067350 1. Sample Protocol 1.A. Vector Cloning 1.Design oligonucleotides: Order oligonucleotide pairs that con
26、tain the desired miRNA target region and, when annealed and ligated into the pmirGLO Vector, result in the miRNA target region in the correct 5 to 3 orientation. Insure that the overhangs created by oligonucleotide annealing are complementary to those generated by restric- tion enzyme digestion of t
27、he pmirGLO Vector in Step 2. Add an internal restriction site to your oligonucleotides for clone confirmation (e.g., NotI in Figure 3 creates a 125bp insert when digested with NotI because of a NotI site at position 93 in the vector). 2.Digest vector: Linearize the pmirGLO Vector with the appropriat
28、e restriction enzymes to generate overhangs that are complementary to the annealed oligonucleotide overhangs. 3.Anneal oligonucleotides: Dilute both oligonucleotides (supplied by user) to 1g/l. Combine 2l of each oligonucleotide with 46l of Oligo Annealing Buffer. Heat at 90C for 3 minutes, then tra
29、nsfer to a 37C water bath for 15 minutes. Use the annealed oligonucleotides immediately, or store at 20C. 1.B. Ligation and Transformation 1.Dilute annealed oligonucleotides 1:10 in nuclease-free water to a final concentration of 4ng/l per oligonucleotide. Ligate 4ng of annealed oligonucleotides and
30、 50ng of linearized vector using a standard ligation protocol. Transform ligated pmirGLO Vector using high-efficiency JM109 competent cells (e.g., Cat.# L2001). 2.Select clones on ampicillin-containing plates, then select clones containing the oligonucleotides by digesting miniprep-purified DNA (e.g
31、., purified using the PureYield Plasmid Miniprep System, Cat.# A1221) using the unique restriction site in the oligonucleotide pair. The purified plasmid DNA can be transfected directly or expanded to generate more DNA. Additional information about annealing, ligation, transformation and oligonucleo
32、tide design can be found in the GeneClip U1 Hairpin Cloning Systems Technical Manual, #TM256, which is available at: 1.C. An Example of Detecting mi-R21 Activity Using the pmirGLO Vector:miR-21 Construct An overview describing the use of the pmirGLO Vector to interrogate endogenous mi-R21 microRNA i
33、s shown in Figure 2. The presence of broadly endogenous microRNA mi-R21 was monitored in HeLa cells. Constructs contained either an exact match to the 21bp mi-R21 target sequence or a mismatched version of that target site (1) as well as PmeI, XbaI and NotI restriction sites (Figure 3; mismatched se
34、quence is in bold italics). Twenty-four hours after transfection with the mi-R21 pmirGLO Vector constructs, cells were analyzed for luciferase activity using the Dual-GloLuciferase Assay System (Cat.# E2920) and a MicroLumatPlus LB96V luminometer (Berthold). Normalized firefly luciferase activity (f
35、irefly luciferase activity/Renillaluciferase activity) for each construct was compared to that of the pmirGLO Vector no-insert control. For each transfection, luciferase activity was averaged from six replicates. 2. Reference 1.Zeng, Y. and Cullen, B.R. (2003) Sequence requirements for micro RNA pro
36、cessing and function in human cells. RNA9 9, 11223. 7841MA pmirGLO Vector (7350bp) Ampr ori Synthetic poly(A) signal hRluc-neo fusion SV40 early enhancer/promoter cer SV40 late poly(A) signal PGK promoter luc2 MCS 7824MA .GCAAG ATCGC CGTGT AATTC TAGTT GTTTA AACGA GCTCG CTAGC CTCGA GTCTA GAGTC GACCT
37、GCAGG. luc2 PmeI DraI 5 EcoICRI Sac I NheI XhoI SbfI SalI AccI XbaI 3 Figure 1. pmirGLO Vector multiple cloning site.Figure 1. pmirGLO Vector multiple cloning site. 7825MA firefly luciferase gene firefly luciferase protein miR-21 target translation stop codon pmirGLO Vector In absence of miR-21 acti
38、vity. In presence of miR-21 activity. Light No firefly luciferase protein No light mRNA destablized; translation blocked Figure 2. Mechanism of action of the pmirGLO Vector.Figure 2. Mechanism of action of the pmirGLO Vector. 7826MB mi-R21 mismatch sense, PmeI and XbaI mi-R21 antisense, PmeI and Xba
39、I mi-R21 sense, PmeI and XbaI mi-R21 mismatch antisense, PmeI and XbaI XbaI 5 AAAC TA GCGGCCGC TAGT TCAACATCAGTCTGATAAGCTA T 3 PmeI NotI internal sitemi-R21 target sequence XbaI 5 AAAC TA GCGGCCGC TAGT TCAACATCAGAAGATAAGCTA T 3 3 TTTG AT CGCCGGCG ATCA AGTTGTAGTCTTCTATTCGAT AGATC 5 3 TTTG AT CGCCGGCG
40、 ATCA AGTTGTAGTCAGACTATTCGAT AGATC 5 PmeI NotI internal sitemi-R21 target sequence Figure 3. Sample oligonucleotides for mi-R21.Figure 3. Sample oligonucleotides for mi-R21. 7827MA 0 50 100 No insertmi-R21mi-R21 mismatch Percent firefly:Renilla luciferase activity compared to no-insert control 100% 5% 78% Figure 4. Normalized luciferase activity using the pmirGLO Vector with an mi-R21 target sequence. Figure 4. Normalized luciferase activity using the pmirGLO Vector with an mi-R21 target sequence.