The kits use the dna rejoining activity of dna topoisomerase i and the dna. Kit recommends not exceeding 3,000 bp inserts and dont use fragments that are too small either. The standard in cloning when it comes to cloning, topo cloning technology has been a reliable partner for thousands of scientists for over ten years. Ligationtopo cloning kit is located in rm 319 on righthand side freezer. Primers are also provided, allowing screening for inserts by colony pcr andor sequencing. Fusion cloning provides rapid and directional cloning into any vector, at any site of linearization, with 95% cloning efficiency. Topo ta cloning kits are designed for cloning pcr products directly from a pcr reaction in just 5 minutes 1. Each kit contains a box with topo ta cloning reagents box 1 and a box with one shot competent e. K457502, refer to the manual supplied with the miniprep kit. The topo ta cloning kit is based on the ta cloning method and uses the pcr2. Jan 22, 2008 since the pentrdtopo cloning kit invitrogen has now become available, only a 4bp cacc leader needs to be added to the 5pcr primer for gene amplification. Kit for sequencing fiveminute cloning of taq polymeraseamplified pcr products for sequencing catalog nos.
This cloning technique utilizes the ability of thymine to hybridize to adenine in the presence of ligases. K451002 and k450002 refer to the manual supplied with. Zero blunt topo pcr cloning kit options the zero blunt topo pcr cloning kit for direct insertion of bluntended pcr products into a plasmid vector can be purchased with a variety of competent cells. Upon rece ipt, store the box es as d etailed below. Digestion of this vector in two sequential reactions with bamh i and xcm i gives a linearized vector with 3t overhangs and a low background of nonrecombinants. Kits supplied with competent cells containa box with zero blunt topo pcr cloning reagents box 1 and a box with one shot e. In this exercise we will simulate topocloning using the plasmid vector pcriitopo thermofisher supplies this vector in linearized form, with covalentlyattached topoisomerase enzyme, and in the tavector case, with 3. K450002 and k451002 are shipped with an additional box containing reagents for plasmid purification box 3. Zero blunt topo pcr cloning kit thermo fisher scientific.
Invitrogen zero blunt topo pcr cloning kit, with pcrblunt. Commercial kits, such as the zero blunt cloning kit from invitrogen, are available. In general, the former option is recommended because it does not require the use of iptg. Bluntend pcr products clone directionally at greater than 90% efficiency. This post focuses on sticky end topo also called topo. Ta cloning kits are available from different manufacturers. Toposiomerase based cloning topo cloning is a dna cloning method that does not use restriction enzymes or ligase, and requires no postpcr procedures. Follow these instructions to use the topo ta cloning kit to clone your pcr product into the topo vector and transform the reaction into chemically. For sticky end topo ta cloning, taq polymerase has a nontemplatedependent terminal transferase activity that adds a single deoxyadenosine a to the 3end of the pcr products.
This allows the pcr product to be directionally cloned into the topo vector to generate an entry clone. Barber lab cloning with invitrogen topo ta cloning kit. Do you need a faster, more reliable solution for dna fragmentation and library. Topo ta cloning kits supplied with the purelink quick plasmid miniprep. Invitrogen zero blunt topo pcr cloning kit, with pcrblunt iitopo vector, one shot top10 chemically competent e. For sticky end topo ta cloning, taq polymerase has a. Download a pdf containing pricing for our full product list. Barber lab cloning with invitrogen topo ta cloning kit this procedure will insert your pcr fragment into a suitable vector in this case pcr2. Thus, the topo technology allows us to easily produce entry clones. Types of topo ta cloning kits two different types of topo ta cloning kits are available in two different. Fiveminute cloning of taq polymeraseamplified pcr products. Fill an icebox with ice before collecting the reagents needed for the topo ta cloning. We have created a circular ta cloning vector, pta plus, based on ppcrscript amp stratagene. The champion pet directional topo expression kits utilize a highly efficient.
The golden gateway cloning system is a combinatorial assembly method that combines golden gate type iis endonucleases cloning with multisite gateway recombination cloning to construct complex. The agilent strataclone pcr cloning kit enables highefficiency, 5minute cloning of pcr products at room temperature. Kit recommends not exceeding 3,000 bp inserts and dont use fragments that are. They use a pcr topo vector with covalently bound topoisomerase i for fast cloning and recombinants. I have a great experience with topo cloning kit and successfully cloned 4 different insert with varying size from 300 to bp. This vector has antibiotic resistance ampicillian and kanamycin as well as having a gene for lacz production. E ach k it co n tain s tw o b o xes as d escribed be low. Invitrogen zero blunt topo pcr cloning kit, with pcrblunt ii. K280020, k280040, k282020, k282040, k283020, k286020, and k286040 a limited label license covers this product see purchaser notification. Incubate at room temperature for at least 5min longer doesnt hurt. Topo cloning is a molecular biology technique in which dna fragments are cloned into specific vectors without the requirement for dna ligases. Other topo cloning kits are available for cloning expand, elongase and other polymerase generatedpcr products, and for the expression of recombinant proteins in e. Ta cloning is a simple and convenient method of subcloning polymerase chain reaction pcr products. Kit contents and storage, continued topo ta cloning reagents topo ta cloning reagents box 1 are listed below.
The kits use the dna rejoining activity of dna topoisomerase i and the dna recombination activity of cre recombinase to yield 95% insert positive clones. The pentrdtopo cloning kits utilize a highly efficient, 5minute cloning strategy topo cloning to directionally clone a bluntend pcr product into a vector for entry into the gateway system or the multisite gateway system. Ta cloning kit for sequencing, per the manufacturers instructions. Cloning technology was invented during the twentieth century and now is poised to help define the twentyfirst. Digestion of this vector in two sequential reactions with bamh i and xcm i gives a linearized vector with 3t overhangs and a low background of nonrecombinants topo ta cloning. Document part number a10605 publication number man0000698 revision a. The technique relies on the basic ability of complementary basepairs adenine a and thymine t to hybridize and form hydrogen bonds. Infusion cloning applications collection takara bio. Its a good thing then that i am a molecular biologist. Topo ta cloning kit for sequencing supplied with the purelink quick plasmid miniprep cat.
We describe a new method, topo vectorligation pcr or tvlpcr. Topo ta cloning home department of molecular biology. The topo xl pcr cloning kit requires a 15minute gel purification step. In the cloning process, the dna is removed from cells, manipulations of the dna are carried out in a testtube, and the dna is subsequently put back into cells. Transform 100 pg1ng of uncut vector to check cell viability, calculate transformation efficiency and verify the antibiotic resistance of the. Invitrogen topo xl pcr cloning kit combines topo cloning, the ccdb gene for positive selection, and a unique gel purification step to enhance cloning of pcr products from 3 to 10 kb. Catalog numbers a11098, a11099, a11100, a11101, and a138.
This vector has antibiotic resistance ampicillian and. Fiveminute cloning of blunt pcr products for expression in insect cells. Each kit contains a box with topo ta cloning reagents box 1 and a box with top10 or top10f one shot competent cells box 2. Types of kits topo ta cloning kits for sequencing are available with either mach1t1r. This flexibility enables a broad range of technologies, from simple cloning through specialized workflows like synthetic gene assembly or highthroughput cloning. Add the components to the tubes in the order as in table 1 below using sterile yellow tips. Kit for sequencing version j 052 250276 topo ta cloning. Topo ta cloning kit, dual promoter, with pcriitopo.
They say a magician is never supposed to tell the secret behind his tricks. The pcrblunt iitopo vector used in this kit comes with lethal ccdb gene for efficient selection of bluntended pcr recombinants products generated by thermostable proofreading polymerases. Dec 02, 20 topo cloning is a molecular biology technique in which dna fragments amplified by taq polymerase are cloned into specific vectors without the requirement for dna ligases. Other topo cloning kits are available for cloning expand, elongase and other polymerase. Topo cloning is a molecular biology technique in which dna fragments are cloned into. This cloning technique utilizes the ability of thymine to.
Pcr cloning vector mix, strataclone pcr cloning kits agilent. For bluntend cloning no taoverhang, dont use taqbased of pcr fragments. We strongly recommend running the following controls during transformations. Almost everyone has heard of dolly, the cloned sheep born in 1996 but what about the. Topo cloning vectors for optimized sequencing molecular info.
In this exercise we will simulate topocloning using the plasmid vector pcriitopo thermofisher supplies this vector in linearized form, with covalentlyattached. Zero blunt topo pcr cloning kit supplied with the purelink quick plasmid miniprep kit cat. Cloning cloning methods ta and topo ta cloning embl. Keep the tube with the topo vector on ice at all times. Selected zero blunt topo pcr cloning kits are available in combo kits combined with a purelink quick plasmid miniprep kit 50 preps for fast plasmid purification of your topocloned inserts for downstream analysis. Pcr cloning kit version k july 24, 2003 250215 zero blunt.
They use a pcrtopo vector with covalently bound topoisomerase i for fast cloning and. Dna cloning cloning is the process of moving a gene from the chromosome it occurs in naturally to an autonomously replicating vector. These controls may help troubleshoot which steps in the cloning workflow has failed. Pcr cloning kits help you get the right clone sooner, freeing up your time to answer more important questions. Kits with competent cells are available with one shot chemically or electrocomp competent cells as described in the following table see page. Here, a protocol using topo ta cloning kit invitrogen is shown. In the cloning process, the dna is removed from cells, manipulations. The neb pcr cloning kit contains a sufficient supply of materials to perform 20 x 10. The topo ta cloning kits for sequencing are shipped on dry ice.
Topoisomerase based cloning topo cloning is a dna cloning method that does not use restriction enzymes or ligase, and requires no postpcr procedures. These controls may help troubleshoot which steps in the cloning workflow has. Topo ta cloning kit for sequencing thermo fisher scientific. Pcr cloning kit fiveminute cloning of bluntend pcr products catalog nos.
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