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