Please try again or contact Customer Service. Trademarks. X65308). A password reset email has been sent to the primary email address associated with your account. 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. Quick PROTOCOL 1 pGEM®-T and pGEM®-T Easy Vector Systems INSTRUCTIONS FOR USE OF PRODUCTS A1360, A1380, A3600 AND A3610. In this study, we describe a method for producing armored L-RNA. The positive samples in this study were termed using the abbreviated name … 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 © 2021 Promega Corporation. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the amplified fragments. A verification email has been sent to the primary email address associated with your account. Summary of Changes The incubation period may be extended to increase the number of colonies after transformation. A resource designed for scientists just embarking on their career, focusing on fundamental technologies and techniques. Complete Protocol Our records indicate that this email address is already registered. We evaluated the cloning efficiency of different size PCR products into three T-vector cloning systems. You have successfully reset your password. Description. Accordingly, as a means of enhancing tissue invasion, tumor cells use matrix metalloproteinases to degrade ECM proteins. Thank you for verifying your email address. 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). You've created a Promega.com account. Please try again or contact Customer Service. Please try again or contact Customer Service. The pGEM®-T Vector was created by linearizing the pGEM®-5Zf(+) Vector with EcoRV at base 51 and adding a T to both 3´-ends. The position of the T is indicated by * in the pGEM®-T Vector Sequence (.txt). 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. We provide medical information and facilitate research collaborations. Reactions using this buffer may be incubated for 1 hour at room temperature. PCR cloning system for expression in mammalian cells. A password reset email has been sent to the primary email address associated with your account. The vector allows preparation of single-stranded DNA due to its f1 Origin of Replication. To protect your privacy, your account has been locked after 6 failed login attempts. 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。 Please contact Customer Service to unlock your account. 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. There was an issue verifying your email address. This page is informational only - this vector is NOT available from Addgene - please contact the manufacturer for further details. pGEM®-T Easy Parental vector for TA cloning of PCR products. We offer numerous convenient solutions to meet your lab's needs. Note: You will not be able to access your account until your email is verified. If initial experiments with your PCR product are suboptimal, ratio optimization may be necessary. The pGEM®-T Easy Vector Systems are convenient systems to clone PCR products generated by certain thermostable polymerases. 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. The pGEM®-T Vector System II contains JM109 Competent Cells in addition to all of the pGEM®-T Vector System I components. Congratulations! Download PDF. Please check your network settings and try again. The pGEM®-T Vector is derived from the pGEM®-5Zf(+) Vector (GenBank® Accession No. Download PDF. Second-generation, high-performance GoTaq® G2 DNA Polymerase in a ready-to-use master mix. There was an issue logging into your account. 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. See Protocol for detailed storage recommendations. pGEM-T easy plasmid DNA (500 ng, Promega, Madison, WI, USA) was then added and incubated for 1 h at 37 °C. A short summary of this paper. There was an issue verifying your email address. The shoot apex tissues of young seedlings were fixed in RNase-free formalin/acetic acid/alcohol fixative. Stay notified of Promega events, products and news. Legal and Trademarks There was an issue with the password reset process. Literature # TM042. Promega Notes 71 , 8–9. However, the in vivo ECM is comprised not only of proteins but also of a variety of non-protein components. RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. Please try again or contact Customer Service. The position of the T is indicated by * in the pGEM®-T Vector Sequence (.txt). Our customer and technical support experts are here to help! PCR cloning vectors with 3 options for insert excision. This is a free resource for the scientific community that is compiled by Addgene.. and Section 5.D. Terms and Conditions You've created a Promega.com account. © 2021 Promega Corporation. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In the current study, we focused on investigating the mechanisms underlying the development of doxorubicin resistance in osteosarcoma.Methods: The human osteosarcoma cell line MG-63 and doxorubicin-resistant MG-63/Dox cells were used in this study. Instructions for Use of Product(s) A1360, A1380, A3600, A3610. You have successfully reset your password. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the amplified fragments. Get in touch with a nearby distributor or sales representative. There was an issue resetting your password. Privacy Policy and Requests for Information 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. Enter your username and we'll send a link to reset your password. 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. 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. 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. Due to our annual Physical inventory orders received after 5PM Central Time on Tuesday March 9 will be shipped according to the following schedule: PDF (548k). This paper. All Rights Reserved. Issai Falcon. Second-generation, high-performance GoTaq® G2 DNA Polymerase with Mg-free buffers. The extracellular matrix (ECM) plays an important role in maintaining tissue homeostasis and poses a significant physical barrier to in vivo cell migration. Download PDF. By creating an account, you confirm that you accept the, Plate Readers, Fluorometers & Luminometers, Privacy Policy and Requests for Information. Insertional inactivation of the alpha-peptide allows recombinant clones to be directly identified by blue/white screening on indicator plates. Instructions for Use of Product(s) A1360, A1380, A3600, A3610. Please request another reset link. Please try again or contact Customer Service. ®Protocol for Ligations Using the pGEM -T and pGEM®-T Easy Vectors and the 2X Rapid Ligation Buffer 3.A. Frackman, S. and Kephart, D (1999) Rapid ligation for the pGEM®-T and pGEM®-T Easy Vector Systems. ... (T. aculeatus and three Zaglossus spp.) Revised 4/17 www.promega.com 2. Thank you for verifying your email address. Molecular Cloning: A Laboratory Manual Third Edition, 1982. Please update your browser to Internet Explorer 11 or above. Canada orders: Ship Monday March 15 for arrival on Tuesday March 16. Legal and Trademarks Molecular Cloning: A Laboratory Manual Third Edition. .. Nicking of DNA was evaluated by ethidium bromide staining after electrophoresis separation in 0.8% agarose gels [ , ]. DNA concentration of linearised recombinant plasmid was determined using the Qubit 1× dsDNA HS Assay Kit (Invitrogen, CA, USA). Please try again or contact Customer Service. Promega Corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, high-value products for the life sciences. 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. Ligation Using 2X Rapid Ligation Buffer 1. Our customer and technical support experts are here to help! Please contact Customer Service to unlock your account. 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. All Rights Reserved. Diese Seite wurde zuletzt am … The pGEM®-T and pGEM®-T Easy Vector Systems include a 2X Rapid Ligation Buffer for ligation of PCR products. This vector is also known as pGEM®‑5Zf(+). Please try again or contact Customer Service. 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. Contains GoTaq® G2 enzyme. PCR products with low concentration or generating heterogeneity in the sequencing chromatograms were cloned into pGEM-T Easy Vector (Promega) for sequencing. Privacy Policy and Requests for Information Let's find the product that meets your needs. There was an issue sending the verification email. We prepared a series of pGEM plasmids (Promega) containing 1, 2, 4, 8, 16, 32, or 64 tandem repeats of the sequence described above. Briefly centrifuge the pGEM ®-T or pGEM -T Easy Vector and Control Insert DNA tubes to collect contents at the bottom of the tube. Yoshino, Y., Ishida, M. and Horii, A. Abstract. Revised 4/17 www.promega.com 2. The insertion site is flanked by BstZI sites. However, ratios of 8:1 to 1:8 have been used successfully. Get in touch with a nearby distributor or sales representative. Introduction. The vector carries the lacZ alpha-peptide and the multiple cloning region arrangement from pUC18 allowing selection of recombinants by blue/white screening. 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. 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. 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. 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. As a result, PCR products amplified using GoTaq® DNA Polymerase will contain A-overhangs which makes it suitable for T-vector cloning. US orders: Ship Saturday March 13 for arrival on Monday March 15. There was an error processing your request. Ratios from 3:1 to 1:3 provide good initial parameters. Product Components and Storage Conditions PRODUCT SIZE CAT. Terms and Conditions ®Briefly centrifuge the pGEM-T or pGEM®-T Easy Vector and Control Insert … The pGEM®-T Vector was created by linearizing the pGEM®-5Zf(+) Vector with EcoRV at base 51 and adding a T to both 3´-ends. The pGEM®-T and pGEM®-T Easy Vector Systems are convenient systems for the cloning of PCR products. Check your inbox to complete email verification. To protect your privacy, your account will be locked after 6 failed attempts. A verified email address is required to access the full functionality of your Promega.com account. Stay notified of Promega events, products and news. Alternatively, a double digestion may be used to release the insert from the vector. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the amplified fragments. However, ratios of 8:1 to 1:8 have been used successfully. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Plates were developed using 1 … We've detected that you are using an older version of Internet Explorer. Please check your network settings and try again. The pGEM®-T Vector cloning region is flanked by recognition sites for the enzyme BstZI. To protect your privacy, your account has been locked after 6 failed login attempts. If initial experiments with your PCR product are suboptimal, ratio optimization may be necessary. www.promega.com Part# TM042 Printed in USA. The 3.9-kb product was cloned into pGEM-T Easy (Promega… 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). There was an issue creating your account. When you select your country, you agree that we can place these functional cookies on your device. Please try again or contact Customer Service. After that, you will need to contact Customer Service to unlock your account. The pGEM®-T Vector Systems are convenient systems to clone PCR products generated by certain thermostable polymerases. Due to our annual Physical inventory orders received after 5PM Central Time on Tuesday March 9 will be shipped according to the following schedule: Download Free PDF. Page 4 Revised 5/07 GGAGA GCTCC CAACG CGTTG GATGC ATAGC TTGAG … X65308). The green and blue arrows indicate 5′-RACE products characterized from WT and 1–910 EagI constructs. Main. Ready-to-use optimized master mix for room-temperature PCR assembly. Note: You will not be able to access your account until your email is verified. Please request another reset link. After that, you will need to contact Customer Service to unlock your account. Dismiss. Using the pGEM-T-mH5 vector that we have previously ... Promega) was added and incubated for one additional hour. Canada orders: Ship Monday March 15 for arrival on Tuesday March 16. Promega Corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, high-value products for the life sciences. Catalog number selected: US orders: Ship Saturday March 13 for arrival on Monday March 15. Download Full PDF Package. 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. The single major product was cloned into the pGEM-T-easy vector (Promega) and was sequenced. 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. Product Components and Storage Conditions PRODUCT SIZE CAT.# pGEM®-7Zf(+) Vector 20µg P2251 The pGEM®-7Zf(+) Vector is provided with a glycerol stock of bacterial strain JM109. Please try again or contact Customer Service. Please try again or contact Customer Service. READ PAPER. The 12/18 version of this Technical Manual was revised to remove references to discontinued products in the notes on sequencing primers in Section 5.B. There was an issue with the password reset process. A verified email address is required to access the full functionality of your Promega.com account. Please try again or contact Customer Service. 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. The pGEM®-T Vector is derived from the pGEM®-5Zf(+) Vector (GenBank® Accession No. 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. Ratios from 3:1 to 1:3 provide good initial parameters. A3600. 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. Check your inbox to complete email verification. Thus, several options exist to remove the desired insert DNA with a single restriction digestion. Ligation Protocol 1. There was an issue logging into your account. 36 Full PDFs related to this paper. Your password reset link has expired. Please try again or contact Customer Service. Instructions for Use of Product(s) The pGEM®-5Zf(+) Vector serves as a standard cloning vector and as a template for in vitro transcription. There was an issue resetting your password. Background: Development of resistance to doxorubicin-based chemotherapy limits its curative effect in osteosarcoma. A verification email has been sent to the primary email address associated with your account. 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. The gold arrows indicate 5′-RACE products characterized from the Δ5G construct. Congratulations! This product is available through the Promega Helix onsite stocking program. You have not verified your email address. 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. Revised 12/18 www.promega.com 3. There was an error processing your request. The high copy number pGEM®-T and pGEM®-T Easy Vectors contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the alpha-peptide coding region of the enzyme beta-galactosidase. 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. A1360, A1380, A3600, A3610. Welcome to Vector Database!. (2007) A new 10-min ligation method using a modified buffer system with a very low amount of T4 DNA ligase: the "coffee break ligation" technique. Enter your username and we'll send a link to reset your password. 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. Our records indicate that this email address is already registered. Insertional inactivation of the α-peptide allows recombinant clones to be directly identified by Blue/White Screening on indicator plates. We provide medical information and facilitate research collaborations. You have not verified your email address. Our website uses functional cookies that do not collect any personal information or track your browsing activity. These samples were collected from Da Longshu village (a, 45 samples), Bai Shiyan village (b, 28 … This vector contains T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the α-peptide coding region of β-galactosidase. # pGEM®-5Zf(+) Vector 20µg P2241 The pGEM®-5Zf(+) Vector is supplied with a glycerol stock of bacterial strain JM109. PLos ONE, Plate Readers, Fluorometers & Luminometers, Save 20% on pGL4 Luciferase Reporter Vectors, enter PGL20 at checkout. The multiple cloning site is flanked by recognition sites for the restriction enzyme BstZI, allowing release of the insert by a single-enzyme digestion. 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. 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. Your password reset link has expired. The insertion site is flanked by BstZI, EcoRI, and NotI sites. Literature # TM042. There was an issue sending the verification email. Our website uses functional cookies that do not collect any personal information or track your browsing activity. https://doi.org/10.1530/JME-17-0142 http://jme.endocrinology-journals.org 2018 Society for Endocrinology Printed in Great Britain Published by Bioscientifica Ltd. Trademarks. Your commerce experience may be limited. 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. Latest generation GoTaq® polymerase—high-performance for your everyday PCR needs. When you select your country, you agree that we can place these functional cookies on your device. There was an issue creating your account. pGEM®-T Parental vector for TA cloning of PCR products. 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. To protect your privacy, your account will be locked after 6 failed attempts. Thus, several options exist to remove the desired insert DNA with a single restriction digestion.