Promega Corporation
2800 Woods Hollow Road Madison, WI 53711-5399USA Telephone 608-274-4330Toll Free 800-356-9526Fax 608-277-2516Internet
www.promega
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Part# 9PIE133
Revised 10/09
Part# 9PIE133
Printed in USA Revised 10/09
pmirGLO Dual-Luciferase miRNA Target Expression Vector:
Cat.#Size E1330
20µg
C a t .# E 1330 c o n t a i n s :P a r t N o .N a m e E133A pmirGLO Vector 20µg C838A Oligo Annealing Buffer
1ml
Description: The pmirGLO Dual-Luciferase miRNA Target Expression Vector (a–e)is designed to quantitatively evaluate microRNA (miRNA) activity by the insertion of miRNA target sites 3´ 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 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 Renilla lucif
erase (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 is 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 luciferase-neomycin resistance cassette (hRluc -neo) is used as a control reporter for normalization of gene expression and stable cell line selection.•Amp r gene allows bacterial selection for vector amplification.
•SV40 late poly(A) signal sequence is positioned downstream of luc2to provide efficient transcription termination and mRNA polyadenylation.•Synthetic poly(A) signal/transcription stop site.
Concentration: 1µg/µl in 10mM Tris-HCl, 1mM EDTA; final pH 7.4.GenBank ®Accession Number:FJ376737.
Storage Conditions:See the storage temperature and expiration date on the Product Information Label.
Functional Assays
Identity Assay: The vector has been sequenced completely and has 100% identity with the published sequence available
at: w
w w w .p r o m e g a .c o m /v e c t o r s /Restriction Digestion:The functional purity of this vector DNA is verified by complete digestion with restriction enzymes at the optimal temperature 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 gel electrophoresis.
Nuclease Assay: Following incubation of 1µg of this vector in Restriction Enzyme Buffer at 37°C for 16–24 hours, no evidence of nuclease activity is detected by agarose gel electrophoresis.Physical Purity:A 260/A 280≥1.80, A 260/A 250≥1.05.
© 2008, 2009 Promega Corporation. All Rights Reserved.
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. Please 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.AF9PI E133 1009E133
(a)READ THIS FIRST BEFORE OPENING PRODUCT
The sale of this product and its use by the purchaser are subject to the terms of a limited use label license, the full text of which is shipped
直接接地箱with this product and also available at: w
w w w .p r o m e g a .c o m \L U L L . That text must be read by the purchaser prior to opening this product to determine whether the purchaser agrees that all use of the product shall be in accordance with the license terms. If the purchaser is not willing to accept the terms of the limited use label license, Promega is willing to accept the return of the unopened product and provide the purchaser with a full refund. However, if the product is opened for any reason, then the purchaser agrees to be bound by the terms of the limited use label license.(b)U.S. Pat. No. 5,670,356.
(c)Australian Pat. No. 2001 285278 and other patents pending.
(d)The method of recombinant expression of Coleoptera luciferase 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 University of California for all other fields) is needed for any commercial sale of nucleic acid contained within or derived from this product.
制卡机
(e)Licensed from University of Georgia Research Foundation, Inc., under U.S. Pat. Nos. 5,292,658, 5,418,155, Canadian Pat. No.2,105,984 and related patents.
S i g n e d b y :
J. Stevens, Quality Assurance
maopPromega Corporation
2800 Woods Hollow Road·Madison, WI 53711-5399 U.S.A. Toll Free in the USA 800-356-9526 Telephone 608-274-4330 www.promega
直链烷基苯
Features List and Map for the pmirGLO Vector
SV40 late poly(A) signal
106–327SV40 early enhancer/promotor
426–844hRluc -neo fusion protein coding region 889–2664Synthetic polyadenylation signal 2728–2776β-lactamase (Amp r ) coding region
3037–3897Col E1-derived plasmid origin of replication 4052–4088Human phosphoglycerate kinase promoter 5094–5609luc2reporter gene
5645–7297Multiple cloning site (MCS, Figure 1)
7306–7350
I.Sample Protocol
A.Vector Cloning
1.Design oligonucleotides: Order oligonucleotide pairs that contain 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 anneali
ng are complementary to those generated by restric-tion enzyme digestion of the 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 appropriate restriction enzymes to
虚拟性成瘾
generate overhangs that are complementary to the annealed oligonucleotide overhangs.3.Anneal oligonucleotides: Dilute both oligonucleotides (supplied by user) to 1µg/µl.
Combine 2µl of each oligonucleotide with 46µl of Oligo Annealing Buffer. Heat at 90°C for 3 minutes, then transfer to a 37°C water bath for 15 minutes. Use the annealed oligonucleotides immediately, or store at –20°C.
B.Ligation and Transformation
1.Dilute annealed oligonucleotides 1:10 in nuclease-free water to a final concentration
of 4ng/µl per oligonucleotide. Ligate 4ng of annealed oligonucleotides and 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., 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 oligonucleotide design can be found in the GeneClip ™ U1 Hairpin Cloning Systems Technical Manual ,
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-R21microRNA is 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 sequence is in italics). Twenty-four hours after transfection with the mi-R21 pmirGLO Vector constructs, cells were analyzed for luciferase activity using the Dual-Glo ®Luciferase Assay System (Cat.# E2920) and a MicroLumatPlus LB96V lumino-meter (Berthold). Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase 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.
II.Reference
1.Zeng, Y. and Cullen, B.R. (2003) Sequence requirements for micro RNA processing
-neo SV40 late PGK ...GCAAG ATCGC CGTGT AATTC TAGTT GTTTA AACGA GCTCG CTAGC
CTCGA GTCTA GAGTC PmeI DraI 5´EcoICRI Sac I
NheI
XhoI SalI AccI
XbaI
3´
F i g u r e 1. p m i r
G L O V e c t o r m u l t i p l e c l o n i n
g s i t
e .
gene firefly luciferase translation In absence of mi-R21activity.
protein
mRNA destablized;F i g u r e 2. M e c h a n i s m o f a c t i o n o f t h e p m i r G L O V e c t o r .
5´ CTAGA TAGCTTATC TT CTGATGTTGA ACTA GCGGCCGC TA GTTT 3´XbaI
mi-R21 mismatch sense, PmeI and XbaI
mi-R21 antisense, PmeI and XbaI
mi-R21 sense, PmeI and XbaI
mi-R21 mismatch antisense, PmeI and XbaI
5´ AAAC TA GCGGCCGC TAGT TCAACATCAG TCT GATAAGCTA T 3´5´ CTAGA TAGCTTATC AGA CTGATGTTGA ACTA GCGGCCGC TA GTTT 3´
5´ AAAC TA GCGGCCGC TAGT TCAACATCAG AA GATAAGCTA T 3´
PmeI NotI internal site
mi-R21 target sequence XbaI
PmeI岩心箱
NotI internal site mi-R21 target sequence F i g u r e 3. S a m p l e o l i g o n u c l e o t i d e s f o r
m i -R 21.
mismatch
P e r c e n t f i r e f l y :R e n i l l a l u c i f e r a s e a c t i v i t y c o m p a r e d t o n o -i n s e r t c o n t r o l
F i g u r e 4. N o r m a l i z e d l u c i f e r a s e a c t i v i t y u s i n g t h e p m i r
G L O V e c t o r w i t h a n m i -R 21 t a r g e t s e q u e n c e .
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