In our experience and the experience of our colleagues, the ta cloning kits from invitrogen carlsbad, ca are the quickest and most robust. Ta cloning a quick method for a tailing pcr products. T overhangs to match the a overhangs of the insert. Briefly centrifuge the pgem t or pgem t easy vector and control insert. The pgemt vector cloning region is flanked by recognition sites for the enzyme bstzi. This method takes advantage of the a overhang added by a pcr enzyme like taq dna polymerase. Restriction enzymes restriction endonuclease promega. It is important to point out, however, that there is nothing wrong with using ta cloning kits from other vendors e. Cloning of taq polymeraseamplified pcr products thermo. They use a pcrtopo vector with covalently bound topoisomerase i for fast cloning and recombinants.
The universal ta cloning method is thus both convenient and laborsaving. Topo ta cloning kits are designed for cloning pcr products directly from a pcr reaction in just 5 minutes 1. Molecular weight markers, or ladders, are a set of standards that are used for determining the approximate size of a protein or a nucleic acid fragment run on an electrophoresis gel. Therefore, ta cloning is especially wellsuited to highthroughput parallel cloning of dna fragments, or dna library construction where additional steps may lead to lower efficiency of clone recovery or loss of library diversity. Specifically, you will first create a recombinant dna molecule by carrying out a ligation reaction, with the goal of connecting a linear dna molecule generated by the polymerase chain reaction with a bacterial plasmid called pgemt. 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.
Most commercial tvectors, such as pgemt easy vector systems promega and pmd tm takara were designed for cloning purposes and a 4 further sub cloning procedure into plasmids with function of. Can pcr products generated with gotaq dna polymerase be used to for t vector cloning. Here, we report a zero background ta cloning system that provides simple and. The pgem t and pgem t easy vectors are linearized vectors with a single 3. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3. Introduction the cloning efficient than bluntended ligation for the cloning of pcrof a dna fragment into a plasmid vector is a routine procedure in recombinant dna technology. Pdf recent advances in universal ta cloning methods for use. In this laboratory, you will carry out a simple cloning experiment in e. The pgemt vector systems are convenient ta vector systems for quickly cloning pcr products. The resultant small rna cdnas are then amplified with a pair of the 5. Promega corporation 2800 woods hollow road madison, wi 537115399 usa. Recent advances in universal ta cloning methods for use in. The pgemt vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below.
Quick protocol 1 pgemt and pgemt easy vector systems instructions for use of products a60, a80, a3600 and a3610. Ta cloning exploits the terminal transferase activity of some dna polymerases such as taq polymerase. Subcloning an introduction to subcloning methods promega. Several years later, in 1993, promega developed pgemt, an evolution of pbr322. May 21, 20 this feature is not available right now. Description continued the pgl4 vector family includes. Get cells out of freezer, thaw on ice for 5 minutes 2.
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. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. Usually clones containing pcr products produce white colonies, but blue colonies can result from pcr fragments that are cloned inframe with the lacz gene. Cloneamp hifi pcr premixhighfidelity pcr for cloning. Ta cloning is a method that ligase is not required and often used when. The pgemt easy vector systems are convenient systems for cloning pcr products. No ligase, postpcr procedures, or pcr primers containing specific sequences are.
Pdf as one of the simplest and most efficient cloning methods. The pgemt vectors are a popular choice for general pcr cloning. One of the easiest methods for cloning bluntended dna fragments including pcr products is tvector cloning, such as with pgemt or pgemt easy vector systems. Basic vectors with no promoter that contain a multiple cloning region for cloning a promoter of. Highfidelity dna polymerases are important for applications in which the dna sequence needs to be correct after amplification. Topo ta cloning kit, dual promoter, with pcriitopo. The pgemt vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. 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 flexi vector system is a directional cloning method for proteincoding sequences. 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. The xcm i digestion of this vector gave rise to a 3.
The pgem t and pgem t easy vector systems are convenient systems for the cloning of pcr products. Ta cloning technology greatly simplifies traditional restriction and ligation cloning with a onestep cloning strategy that eliminates the need for any enzymatic modifications of the pcr product and not require the use of primers that contain restriction enzyme sites. For zebata cloning, the ligation was carried out using the t4 dna ligase from promega catalog no. The cdnas can be cloned in a ta cloning vector for manual sequencing a. Its unique structure, a novel pyrococcuslike enzyme.
Subcloning is a basic procedure in molecular biology for transfer of dna inserts from one vector to another to gain functionality to study the sequence of interest. The technique relies on the ability of adenine a and thymine t complementary base pairs on different dna fragments to hybridize and, in the presence of ligase become ligated together. Ta cloning failure with pgemt easy vector and jm109. Jan 08, 2020 a subset of promega restriction enzymes offer capabilities that include rapid digestion of dna in 15 minutes or less, ability to completely digest dna directly in the gotaq green master mix, and bluewhite cloning qualification which allows for rapid, reliable detection of transformants. Cloning pcr products with pgemt and pgemt easy vectors. Ta cloning also known as rapid cloning or t cloning is a subcloning technique that avoids the use of restriction enzymes and is easier and quicker than traditional subcloning. 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. However, several problems have been encountered using. Excerpts and links may be used, provided that full and clear credit is given to promega corporation with appropriate and specific direction to the original content.
Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to address both demands for. Promega supplies quality, performancetested restriction enzymes for restriction enzyme digestion and cloning needs. They offer all of the advantages of the pgemt vector systems with ecori and noti sites flanking the insertion site. Shop online for a wide selection of promega gotaq dna polymerase taq dna polymerase. Cloneweaver helps you build a customized cloning kit with all of the items your cloning scheme requires. Set up the following reaction in a microcentrifuge tube on ice. This cloning technique utilizes the ability of thymine to hybridize to adenine in the presence of ligases. Simvector makes it so easy to design ta cloning experiments. Ligation protocol with t4 dna ligase m0202 protocols. Incubate at room temperature for 1 hour or overnight at 4c 3.
We evaluated the cloning efficiency of different size pcr products into three tvector cloning systems. This enzyme adds a single, 3a overhang to each end of the pcr product. The pgemt vector system is a convenient system for the cloning of pcr products, designed to improve the efficiency of ligation of the pcr product into the plasmid. Ta cloning is a simple and convenient method of subcloning polymerase chain reaction pcr products. Stay notified of promega events, products and news. The pgem t and pgem t easy vector systems are supplied with 2x rapid ligation buffer. Ta cloning relies on the complementary bases of adenine a and thymine t on different dna fragments to hybridize. Sequence and multicloning site of the pgemt easy vector. If you are using a proofreading polymerase, add a bit of normal taq to the mixture as taq adds the terminal as.
Topo ta cloning topo ta cloning provides a highly efficient, 5minute, onestep cloning strategy topo cloning for the direct insertion of. Our teams are in regular contact with suppliers and distributors worldwide to manage inventory of raw materials to ensure continued availability. My name is robert deyes and i work with promegas technical support. L containing 50 ng of tvector and the corresponding volume of pcr product. These toverhangs at the cloning site improve the efficiency of ligation of pcr products that contain daoverhangs. The dna ligation kit, mighty mix is a 2x ligation premix for highefficiency ligations, especially for bluntended ligations. Description the pgem t and pgem t easy vector systemsa,b are convenient systems for the cloning of pcr products. Provided with green and colorless reaction buffers. Comparing cloning efficiency of the pgemt and pgemt easy vectors to the topo ta cloning vectors. This product introduced a new technique based on ta cloning. This pcr cloning kit contains an optimized 2x cloning master mix with a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for background colony suppression to give a low background. Cloning methods can be divided into two classes, depending on. Pdf recent advances in universal ta cloning methods for. Alternatively, a doubledigestion may be used to release the insert from either vector.
T vectors are linearized plasmids that have been treated to add 3. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. The technique relies on the ability of adenine a and thymine t complementary basepairs on different dna fragments to hybridize and, in the presence of ligase, become ligated together. Systems are convenient ta vector systems for quickly cloning pcr products. 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. In this vector, suppose if your insert does not have a smai site, they you. Pdf recent advances in universal ta cloning methods for use in.
This dna ligation kit can also be used for ligation into ta vectors. The 2x master mix contains enzyme, optimized buffer, and dntps, allowing rapid setup of pcr reactions and facilitating highthroughput applications for multiple cloning samples. Tvector cloning, or ta cloning, is a convenient method for cloning pcr products generated with taq dna polymerase. Instructions for use of products a60, a80, a3600, a3610. Cloneamp hifi pcr premix is designed for use with the infusion cloning system due to its exceptionally accurate and efficient dna amplification. Construction of a high efficiency pcr products cloning t. Promega corporation 2800 woods hollow road madison, wi 537115399. The pgemt easy vector systems are convenient systems for cloning pcr. A protocol for cloning pcr products using t vectors. These standards contain predetermined fragment or protein sizes and concentrations. Each promega pcr cloning system is provided with a control insert. 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. No ligase, postpcr procedures, or pcr primers containing specific sequences are required.
How to clone a pcr amplified product in a pet expression vector. Purchase your selection instantly, save it or email it to a purchasing agent. Essentially all subcloning reactions proceed the same way as illustrated in the figure below. Ta cloning is a method that ligase is not required and often used when constructing cloning vectors. Ta cloning is a subcloning technique that doesnt use restriction enzymes and is easier and quicker than traditional subcloning.
A versatile zero background tvector system for gene cloning and. Topo ta cloning provides a highly efficient, 5minute, onestep cloning strategy topo cloning for the direct insertion of taq polymeraseamplified pcr products into a plasmid vector. The ta cloning wizard accepts both commercial tvectors or a restriction designed tvector to combine them with the modified pcr product and generates a highly accurate recombinant dna plasmid map. Ta cloning failure with pgemt easy vector and jm109 supercompetent cells from. How to clone a pcr amplified product in a pet expression.
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