X65308). Instructions for Use of Product(s) A1360, A1380, A3600, A3610. The pGEM®-5Zf(+) Vector serves as a standard cloning vector and as a template for in vitro transcription. Description. We have successfully cloned PCR products generated using GoTaq® and GoTaq® Flexi DNA Polymerases into the pGEM®-T (Cat.# A3600), pGEM®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors. In this study, we describe a method for producing armored L-RNA. The Promega mission statement is: To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. Please try again or contact Customer Service. Shop Now ›, Rapid Ligation for the pGEM®-T and pGEM®-T Easy Vector Systems, Comparing Cloning Efficiency of the pGEM®-T and pGEM®-T Easy Vectors to the TOPO TA Cloning® Vectors, Shorten the Ligation Time for the pGEM®-T Vector Systems, TRE5-A retrotransposition profiling reveals putative RNA polymerase III transcription complex binding sites on the, Privacy Policy and Requests for Information, Insert excision with a BstZI single digest, Ligation can be completed in 1 hour at room temperature, Available with or without competent cells. All Rights Reserved. Your commerce experience may be limited. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the amplified fragments. Check your inbox to complete email verification. Alternatively, a double digestion may be used to release the insert from the vector. The pGEM®-T Easy Vector Systems are convenient systems to clone PCR products generated by certain thermostable polymerases. These samples were collected from Da Longshu village (a, 45 samples), Bai Shiyan village (b, 28 … The vector allows preparation of single-stranded DNA due to its f1 Origin of Replication. We've detected that you are using an older version of Internet Explorer. Ligation Protocol 1. Introduction. Get in touch with a nearby distributor or sales representative. In addition, we excised the gene encoding GFP-mRNA-96-mer from pTRE-GFP-96-mer and inserted it into plasmid pGEM, because that plasmid contains a bacteriophage T7 promoter. Catalog number selected: Promega Corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, high-value products for the life sciences. DNA concentration of linearised recombinant plasmid was determined using the Qubit 1× dsDNA HS Assay Kit (Invitrogen, CA, USA). The pGEM-T Vector is prepared by cutting Promega's pGEM-5Zf(+) Vector with EcoR V and adding a 3' terminal thymidine to both ends.These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid. Please check your network settings and try again. Note: You will not be able to access your account until your email is verified. Diese Seite wurde zuletzt am … Complete Protocol Thus, several options exist to remove the desired insert DNA with a single restriction digestion. The green and blue arrows indicate 5′-RACE products characterized from WT and 1–910 EagI constructs. One of the easiest methods for cloning blunt-ended DNA fragments including PCR products is T-vector cloning, such as with pGEM®-T or pGEM®-T Easy Vector Systems.This method takes advantage of the “A” overhang added by a PCR enzyme like Taq DNA Polymerase.T vectors are linearized plasmids that have been treated to add 3′ T overhangs to match the A overhangs of the insert. The positive samples in this study were termed using the abbreviated name … The pGEM®-3Zf(+) and pGEM®-3Zf(–) Vectors contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the α-peptide coding region of β-galactosidase. Download Free PDF. We evaluated the cloning efficiency of different size PCR products into three T-vector cloning systems. Terms and Conditions By creating an account, you confirm that you accept the, pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual, pGEM T and pGEM T Easy Vector Systems FB033, 2017 Please check your network settings and try again. Please try again or contact Customer Service. Legal and Trademarks PCR products were gel-purified, cloned into the pGEM-T Easy Vector system (Promega Corporation, WI, USA) and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Yoshino, Y., Ishida, M. and Horii, A. Instructions for Use of Product(s) A1360, A1380, A3600, A3610. Our website uses functional cookies that do not collect any personal information or track your browsing activity. There was an issue resetting your password. Trademarks. www.promega.com Part# TM042 Printed in USA. We provide medical information and facilitate research collaborations. Canada orders: Ship Monday March 15 for arrival on Tuesday March 16. In this study, a specific 277-bp cDNA fragment of DHD4 was amplified and then cloned into the pGEM-T Easy vector (Promega), which was used to produce antisense and sense RNA probes. When you select your country, you agree that we can place these functional cookies on your device. When you select your country, you agree that we can place these functional cookies on your device. Frackman, S. and Kephart, D (1999) Rapid ligation for the pGEM®-T and pGEM®-T Easy Vector Systems. PCR cloning vectors with 3 options for insert excision. There was an issue creating your account. The pGEM®-11Zf(+) Vector is a standard cloning vector that contains T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the α-peptide coding region of β-galactosidase. Get in touch with a nearby distributor or sales representative. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the amplified fragments. See Protocol for detailed storage recommendations. Die In-vitro-Transkription, also die DNA-abhängige Synthese von RNA im Reagenzglas, ist eine molekularbiologische Methode zur Erzeugung von RNA und zur Untersuchung von Promotoren und ihrer Aktivierung durch Transkriptionsfaktoren. Legal and Trademarks A verification email has been sent to the primary email address associated with your account. There was an issue logging into your account. If initial experiments with your PCR product are suboptimal, ratio optimization may be necessary. 36 Full PDFs related to this paper. The insertion site is flanked by BstZI sites. We provide medical information and facilitate research collaborations. The pGEM ®-T and pGEM ®-T Easy Vector Systems are convenient systems for the cloning of PCR products.The vectors are prepared by cutting the pGEM ®-5Zf(+) and pGEM ®-T Easy Vectors, respectively, with EcoR V and adding a 3´ terminal thymidine to both ends. Please try again or contact Customer Service. Congratulations! Ratios from 3:1 to 1:3 provide good initial parameters. Congratulations! Please update your browser to Internet Explorer 11 or above. The assay sensitivity was determined using pGEM-T Easy Vector plasmids (Promega, Madison, WI) containing the target sequence of the N (961 bp) and S (1119 bp) genes of SARS-CoV-2. Revised 4/17 www.promega.com 2. A1360, A1380, A3600, A3610. Download PDF. The pGEM®-T Easy Vector multiple cloning region is flanked by recognition sites for the restriction enzymes EcoRI, BstZI and NotI, providing three single-enzyme digestions for release of the insert. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the amplified fragments. Please contact Customer Service to unlock your account. These single 3´-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmids by preventing recircularization of the vector and providing a compatible overhang for PCR products generated by certain thermostable polymerases. This paper. Please request another reset link. ®Protocol for Ligations Using the pGEM -T and pGEM®-T Easy Vectors and the 2X Rapid Ligation Buffer 3.A. The pGEM®-T Easy Vector Systems offer all of the advantages of the pGEM®-T Vector Systems with the added convenience of recognition sites for BstZI, EcoRI and NotI flanking the insertion site. The pGEM®-3Z Vector is intended for use as a standard cloning vector, as well as for the highly efficient synthesis of RNA in vitro. This product is available through the Promega Helix onsite stocking program. The pGEM®-T Vector is ready to use in ligation reactions, prepared by cutting the pGEM®-5Zf(+) Vector with EcoRV and adding a 3´ terminal thymidine to both ends. A3600. Our customer and technical support experts are here to help! Our customer and technical support experts are here to help! .. Nicking of DNA was evaluated by ethidium bromide staining after electrophoresis separation in 0.8% agarose gels [ , ]. Please try again or contact Customer Service. However, ratios of 8:1 to 1:8 have been used successfully. pGEM®-T Easy Vector Systemは、従来のpGEM®-T Vector Systemの機能に加え、マルチクローニングサイトの両端にEcoRIとNotIサイトが加えられました。そのため、1種類（NotI、EcoRIあるいはBstZI）の制限酵素を用いるだけで、クローニング後のインサートDNAを簡単に切り出すことがきます。 A verified email address is required to access the full functionality of your Promega.com account. To protect your privacy, your account will be locked after 6 failed attempts. Please try again or contact Customer Service. In the pGEM®-T Vector, T7 and SP6 RNA polymerase promoters flank a multiple cloning region within the α-peptide coding region for β-galactosidase. Promega Notes 71 , 8–9. These single 3´-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid by preventing recircularization of the vector and providing a compatible overhang for ligation of PCR products with A overhangs. The pGEM ®-T and pGEM -T Easy Vector Systems have been optimized using a 1:1 molar ratio of the Control Insert DNA to the vectors. The pGEM®-T and pGEM®-T Easy Vector Systems gave a high number of recombinants across a broad range of insert sizes (0.5–3kb) while the TOPO TA Cloning® system worked well for inserts less than 1kb, but showed a striking decrease in performance with larger insert sizes (1–3kb). There was an error processing your request. However, the in vivo ECM is comprised not only of proteins but also of a variety of non-protein components. Pod pepper (Capsicum frutescens) is widely planted in China, especially around Wenshan city, Yunnan province, and viral diseases have now also become a major threat to pepper production in Yunnan.During July 2019, 89 pepper leaf samples were collected from three different fields in Wenshan. Background: Development of resistance to doxorubicin-based chemotherapy limits its curative effect in osteosarcoma. You have successfully reset your password. The pGEM®-T Vector is ready to use in ligation reactions, prepared by cutting the pGEM®-5Zf(+) Vector with EcoRV and adding a 3´ terminal thymidine to both ends. Second-generation, high-performance GoTaq® G2 DNA Polymerase in a ready-to-use master mix. There was an issue logging into your account. You have successfully reset your password. The pGEM ®-T and pGEM -T Easy Vector Systems have been optimized using a 1:1 molar ratio of the Control Insert DNA to the vectors. pGEM-T easy plasmid DNA (500 ng, Promega, Madison, WI, USA) was then added and incubated for 1 h at 37 °C. Thus, several options exist to remove the desired insert DNA with a single restriction digestion. There was an issue with the password reset process. US orders: Ship Saturday March 13 for arrival on Monday March 15. The pGEM®-T Vector System II contains JM109 Competent Cells in addition to all of the pGEM®-T Vector System I components. One of the easiest methods for cloning blunt-ended DNA fragments including PCR products is T-vector cloning, such as with pGEM®-T or pGEM®-T Easy Vector Systems.This method takes advantage of the “A” overhang added by a PCR enzyme like Taq DNA Polymerase.T vectors are linearized plasmids that have been treated to add 3′ T overhangs to match the A overhangs of the insert.
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