After pcr using taq polymerase, the fragments are cloned into plasmids by ta cloning taq amplified for sequencing. Alternatively, a doubledigestion may be used to release the insert from either vector. Cloneweaver helps you build a customized cloning kit with all of the items your cloning scheme requires. Nanobret e3 ligase interaction assays kit available through custom assay services. So if you are starting a new project, we are with you every step of the way. A protocol for cloning pcr products using t vectors. The pgemt easy vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases.
Purchase your selection instantly, save it or email it to a purchasing agent. No ligase, postpcr procedures, or pcr primers containing specific sequences are. Can pcr products generated with gotaq dna polymerase be used to for t vector cloning. Bolivar developed pbr322 in 1977, the first cloning vector that paved the. Each ligation reaction contained fmol of a pt7 blue tvector and 39 fmol of a 2.
There is no genbank accession number for the pgemt easy vector. Ta cloning exploits the terminal transferase activity of some dna polymerases such as taq polymerase. The pgem t and pgem t easy vector systems are convenient systems for the cloning of pcr products. The parent vector is linearized at the position indicated by in this pgemt easy vector sequence and a t is added at each end. Includes an array of cloning and subcloning vectors, and the convenient flexi cloning systems for protein expression.
Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to address both demands for. Once you have designed the cloning scheme, gathering all of the required reagents to get you from construct to expression and analysis is not trivial. Find ligases, polymerases, pnk, phosphatases, nucleases and more. This makes it possible to clone this pcr product directly into a linearized cloning vector with single, 3t overhangs. Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to address both demands for diagnostic tools and reliable delivery of all products as quickly as possible. Quick protocol 1 pgemt and pgemt easy vector systems instructions for use of products a60, a80, a3600 and a3610. They offer all of the advantages of the pgemt vector systems with the added convenience of recognition sites for ecori and noti flanking the insertion site. Cloning system and protein expression vectors functional protein analysis usually requires recombinant expression of the protein of interest. The 500bp fragment is present at increased intensity for easy identification. Yes you can insert any gene sequence in pgemt easy vector but one thing you have. Instructions for use of products a60, a80, a3600, a3610. Incubate at room temperature for 1 hour or overnight at 4c 3. If you are using a proofreading polymerase, add a bit of normal taq to the mixture as taq adds the terminal as.
Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect contents at the bottom of the tube. It is based on two rarecutting restriction enzymes, sgfi and pmei, and provides a rapid, efficient and highfidelity way to transfer proteincoding regions without the need to resequence. There are a multitude of these and especially since you are cloning using a promega kit, i would suggest using gotaq. A versatile zero background tvector system for gene cloning and. Promega supplies quality, performancetested restriction enzymes for restriction enzyme digestion and cloning needs. Tavector ligation efficiency of takarabrand dna ligation kit, mighty mix and similar kits from three competitors tavector ligation efficiency of takarabrand dna ligation kit, mighty mix and similar kits from three competitors. In addition, the vector contains both the sp6 and t7 rna. The size of the pcr product has a limitation of cloning into pgemteasy vectors. April, 1998pcr based cloning kits table the end table pdf format pcr has found applications in almost every imaginable facet of molecular biology, and for many applications, looking at a band on a gel is not enough. Because it is difficult to generate a highquality ta cloning vector in individual. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak.
The pgemt and pgemt easy vector systemsa,b facilitate the cloning of pcrc products by providing linear vectors that have a single thymidine extension. Additionally, any other dna fragment, either blunt or stickyend, can be. The pgemt vector cloning region is flanked by recognition sites for the enzyme bstzi. These vectors are ready to use in ligation reactions. Choose from a large catalog of restriction enzymes, including a subset of enzymes that are capable of rapid digestion of dna in 15 minutes or less. Tacloning vector from a specially designed parent plasmid.
Cloning vectors and kits the cloning of a specific fragment of dna into a vector such as a plasmid is one of the foundational steps that enables additional characterization of that sequence. The pgem t and pgem t easy vector systems are supplied with 2x rapid ligation buffer. The t7 and sp6 promoters allow in vivo or in vitro transcription of the insert to produce sense or antisense products. The pgemt easy vector systems are convenient systems for cloning pcr. Get cells out of freezer, thaw on ice for 5 minutes 2. For ta cloning, it is optimal if the pcr primers have gs at the 5 end as this will maximize the probability of taq polymerase adding the terminal a overhang see brownstein et al 1996. The 100bp dna ladder has eleven fragments that range in size from 100bp to 1,000bp in 100bp increments with an additional band at 1,500bp. The pgemt easy vector systems are convenient systems to clone pcr products. Eliminates any enzymatic modifications of the pcr product. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. Vectors such as the pgem series contain multiple cloning sites to excerpt fragments generated by digestion with specific restriction enzymes.
Ta cloning is a technique that avoids the use of restriction enzymes and is thus easier and faster than traditional subcloning. In addition, the vector contains both the sp6 and t7 rna polymerase promoters flanking the multiple cloning region. The ligafast rapid dna ligation system is designed for the efficient ligation of stickyended dna inserts into plasmid vectors in just 5 minutes bluntended inserts in as little as 15 minutes. Ta cloning failure with pgemt easy vector and jm109 supercompetent cells from promega. You can even save your oneofakind cloning kit for future endeavors. Application have included the cloning of small pcr products for sequencing 100300 bp and larger pcr fragments generated from genomic dna or cdna 5005000 bp. Promega corporation 2800 woods hollow road madison, wi 537115399. Ta cloning failure with pgemt easy vector and jm109. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. The pgemt vectors are a popular choice for general pcr cloning. The pgemt vector systems are convenient ta vector systems for quickly cloning pcr products.
The pgemt vector systems are convenient ta vector systems for quickly cloning. Usually, this is done for the pcramplified dna after round 7 or later selection rounds, depending on the enrichment of the binders. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3. Sequencing, expressing, mutatingall require cloning. Tvector cloning, or ta cloning, is a convenient method for cloning pcr products generated with taq dna polymerase. This enzyme adds a single, 3a overhang to each end of the pcr product. The pgemt and pgemt easy vectors are linearized vectors with a single. For this purpose, the protein coding sequence is cloned into a suitable expression vector and transferred into cells. Can the cloning kit be used for inserts that are not necessarily pcr amplicons. T%20and%20pgemt%20easy%20vector%20systems%20protocol.
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