Topo technology enables inserts with compatible ends to be readily joined to the vector in 5 minutes, without the need for additional ligation steps. Invitrogen topo ta cloning kit for sequencing, without. I did topo cloning yesterday and spread the bacteria onto lb ampicillin 50 ugml and culture at 37 over night. Bsaisite removed from the ampicillinresistant gene allows for the cloning of golden gate assembly modules fast cloning experiments with 5minute ligation step. Information is provided elsewhere in the manual if you need help with any of the steps. Incubate at room temperature for at least 5min longer doesnt hurt.
The ta topo cloning technique relies on the ability of adenine a and. Topo cloning is a molecular biology technique in which dna fragments are cloned into specific vectors without the requirement for dna ligases. I am using a topota cloning kit from thermo fisher k4575j10 for cloning. Instruction manual pbad topo ta expression kit fiveminute cloning of taq polymeraseamplified pcr products for regulated expression in e. Oct 22, 2019 i am using a topota cloning kit from thermo fisher k4575j10 for cloning. Review and cite ta cloning protocol, troubleshooting and other methodology information contact experts in ta cloning to get answers. Topo ta cloning kit manual invitrogen by eliminating timeconsuming and tedious restriction site cloning, topo cloning topo ta cloning kit for subcloning, with top10 e. In this approach, target dna molecules and cloning vector are generated by pcr with 610 bases of homology between the neighboring fragments. The topo ta cloning method combines the advantages of ta cloning with the ligation activity of topoisomerase i. Make a good pcr reaction in 50 l volume with special topoprimers. User manual pef6v5 his topo ta expression kit fiveminute cloning of taq polymerase amplified pcr products for highlevel expression in mammalian cells catalog no.
K457502 is shipped with an additional box containing reagents for plasmid purification box 3. The agilent easya highfidelity pcr cloning enzyme is a proprietary thermostable dna polymerase formulation specifically designed for improved cloning with the strataclone pcr cloning kit or with the topo ta cloning vector and other tuvectors. V5 topo ta cloning kit contains the topo adapted virapower hiperform lentiviral expression vector, plenti6. Topo cloning utilizes the taq polymerase which naturally leaves a single adenosine a overhang on the 3. Before starting determine a strategy for pcr pages 34 and generate the pcr product containing your gene of interest page 5. Topo cloning utilizes the vacinnia dna topoisomerase i enzyme, which functions both as a restriction enzyme and as a ligase. In this tutorial we will learn how to clone human proinsulin, the precursor of insulin, into topo vectors using ta, blunt and directional methods. K4575j10, k457501, k457540, k458001, k458040, k459501, k459540 a limited label license covers this product see purchaser notification. It has once again established its primacy as the molecular laboratory manual and is likely to be found on lab benches. The user friendly dna engineering method allows combining multiple pcr fragment assembly, nucleotide sequence alteration and directional cloning.
Proofreading enzyme high cloning efficiency of taq. Then i repeated the same steps cloning my purified vector into the. Jan 02, 2007 since the development of the first generic cloning and sequencing topo ta cloning kit, invitrogen has expanded the system with a multitude of vectors for various downstream cloneready functions e. Item concentration amount 10 reactions amount 20 reactions amount 40 reactions pcr4topo 25 10 ng l plasmid dna in.
Types of topo ta cloning kits two different types of topo ta cloning kits are available in two different. The consideration paid for cloning technology products e. The ta topo cloning technique relies on the ability of. Topo cloning procedure for experienced users introduction this quick reference sheet is provided for experienced users of the topo cloning procedure. A bp cloning, b topo cloning, and c restriction enzyme and ligase cloning.
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. I thought i should topo ta clone these amplicons, but. Topo ta cloning kits are available from invitrogen. The field below shows which of the selected sequences have been detected as vectors, all other sequences are inserts. Cloning of pcr products into topo ta vectors following pcr amplification with the enzyme taqpolymerase, a single deoxyadenosine da is added to the 3 ends of pcr products by the enzymes terminal transferase activity. Zero blunt topo pcr cloning kits are shipped on dry ice. The topoisomerase will join the pcrproduct into the vector and cut itself out. Three different options ta blunt or directional cloning are shown on the top.
If directional is selected the user can define an overlap sequence. Not intended for any animal or human therapeutic or diagnostic use. K451002 and k450002 refer to the manual supplied with the miniprep kit. Cloning cloning methods ta and topo ta cloning embl. Item concentration amount 10 reactions amount 20 reactions amount 40 reactions pcr4 topo 10 ngl plasmid dna in. V5 topo for quick pcrbased cloning and highlevel expression of a target gene in dividing and nondividing mammalian cells. Shop online for a wide selection of invitrogen topo ta cloning kit for sequencing, without competent cells provides a highly efficient, 5minute, onestep cloning strategy. No ligase, postpcr procedures, or pcr primers containing specific sequences are required. K430001, k430040 version n april 2004 250196 a limited use label license covers this product see purchaser notification. Ligationtopo cloning kit is located in rm 319 on righthand side freezer. Quick and efficient pcr cloning with topo ta cloning.
Taq dna polymerase catalyzes the nontemplate directed addition of an adenine residue to the 3. Item concentration amount 10 reactions amount 20 reactions amount 40 reactions pcr4topo 10 ngl plasmid dna in. 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. The topo ta cloning kits for sequencing are shipped on dry ice. However, a lot of users still stick with the original topo ta cloning kit with the pcr2. Kits supplied with competent cells contain a box with zero blunt topo pcr cloning reagents box 1 and a box with one shot e. Dec 02, 20 topo vectors are designed in such a way that they carry this specific sequence 5. 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. Kit for sequencing fiveminute cloning of taq polymeraseamplified pcr products for sequencing catalog nos. A laboratory manual fills the same niche in the laboratory with information to help both the inexperienced and the advanced user. Topo cloning toposiomerase based cloning, often called topo cloning or ta cloning, is a method that relies on the hybridization of the complementary base pairs adenine a and thymine t.
The three possible methods that lead to the entry clone are depicted. This allows direct ligation of pcr products in just 5 minutes. Topo ta cloning kit for sequencing supplied with the purelink quick plasmid miniprep cat. Topo vector 1ul pcr product 4ul salt solution 1ul total 6 ul, room temperature let react for 30 min. If this field is blank it has the same effect as blunt cloning. Clone pcramplified dna fragments blunt or aoverhang directly into a choice of over 40 subcloning, sequencing, or expression vectors in just 5 minutesand obtain up to 95% recombinant clones. Updated to allow for in vitro transcription with both sp6 and t7 promoters. Kits containing competent cells include box with topo ta cloning reagents box 1 and a box with one shot competent. Invitrogen topo pcr cloning technology was developed to help improve cloning efficiency, simplify protocol setup, and accommodate a wide range of pcr insert sizes. Ligation topo cloning kit is located in rm 319 on righthand side freezer. Topo ta cloning reagents topo ta cloning reagents box 1 are listed below. Zero blunt topo pcr cloning kit supplied with the purelink quick plasmid. Cloning for the direct insertion of taq polymeraseamplified pcr products into a plasmid vector.663 264 326 930 793 1443 492 1369 559 1140 1462 988 453 857 139 327 669 409 1080 346 1148 837 1322 163 602 1038 930 1442 1321 277 1393 818 460 1076 1425