No ligase, postpcr procedures, or pcr primers containing specific sequences are required. Left plate serves as the control, with vector backbone only, right plate contains pcr. Invitrogen ta cloning kits rely on the complementary bases of adenine a and thymine t on different dna fragments to hybridize together. 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.
Barber lab cloning with invitrogen topo ta cloning kit. 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. I performed the ta cloning and plated the cells at the same time protocol. The ta cloning method can be easily modified so that the same tvector can be used to clone any doublestranded dna fragment, including pcr products amplified by any dna polymerase, as well as all blunt and stickyended dna species. Ligation protocol with t4 dna ligase m0202 protocols.
You should use this protocol in any the following circumstances. The principle behind the ta cloning kit is quite simple. Perform pcr reaction as normal using pfu polymeraseif did pcr using taq skip to step 4. I amplified my pcr products by using econotaq polymerase kit, then purify it using promega wizard purification kit. I performed the ta cloning and plated the cells at the same time. The procedure exploits the terminal transferase activity of certain thermophilic dna polymerases, including thermus aquaticus taq polymerase. Centrifuge vials containing the ligation reactions briefly and place them on ice. Given its importance it is remarkable that cloning strategies for many of the popular dna components are not standardised.
Invitrogen 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. Topo ta cloning kit for sequencing supplied with the purelink quick plasmid miniprep cat. Cloning cloning methods ligation independent cloning lic. Quick protocol 1 pgemt and pgemt easy vector systems instructions for use of products a60, a80, a3600 and a3610. Consider the following factors when choosing the protocol that best suits your. The dna ligation kit, mighty mix is a 2x ligation premix for highefficiency ligations, especially for bluntended ligations. Ta cloning is a simple and convenient method of subcloning polymerase chain reaction pcr products. This post focuses on sticky end topo also called topo. May 27, 2015 this video explains how invitrogen topo pcr cloning technology works. We have used the ta cloning kit extensively in our lab for cloning various genes and gene fragments. K202020 and k202040 are not supplied with one shot competent cells. Barber lab cloning with invitrogen topo ta cloning kit this procedure will insert your pcr fragment into a suitable vector in this case pcr2. Lic is a cloning method that makes use of annealing of singlestranded complementary overhangs on the target vector and a pcrgenerated insert of at least 12. The pgemt vectors are a popular choice for general pcr cloning.
The technique relies on the basic ability of complementary basepairs adenine a and thymine t to hybridize and form hydrogen bonds. For example, ta cloning uses regular taq dna polymerase to add a single 3a overhang to the ends of the pcr product. Invitrogen topo ta cloning kit for subcloning, without. Ta cloning is one of the simplest forms of cloning.
If you are using a proofreading polymerase, add a bit of normal taq to the mixture as taq adds the terminal as. Kits containing competent cells include box with topo ta cloning reagents box 1 and a box with one shot competent. Dec 12, 2015 this lecture explains about ta cloning process in details. First,i performed pcr reaction for 4 different genes using each gene specific primers. Two protocols are provided to transform one shot chemically competent e. Nov 06, 2003 the ta cloning kit from invitrogen proves a highly efficient method of cloning a pcr product into a plasmid vector in one step. 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. 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. The yield of my purified pcr products is about 100 ng. Invitrogen topo ta cloning kit for sequencing, without competent cells provides a highly efficient, 5minute, onestep cloning strategy topo cloning for the direct insertion of taq polymeraseamplified pcr products into a plasmid vector for sequencing.
Oct 27, 2016 toposiomerase based cloning topo cloning is a dna cloning method that does not use restriction enzymes or ligase, and requires no postpcr procedures. In conclusion, this is a very useful kit for cloning pcr products which invitrogen has continued to improve since its release. The manual is clearly written and very easytofollow. If screening of colonies containing plasmid with insert dna is necessary, we recommend doing. Invitrogen gateway cloning technology has been cited by life science researchers more than 2,000 times. Invitrogens ta cloning kit dual promoter biocompare. Ta cloning kits for sequencing are shipped on dry ice. Here, a protocol using topo ta cloning kit invitrogen is shown.
Ta cloning topo ta cloning provides a highly efficient, 5minute, onestep cloning strategy topo cloning for the direct insertion of. Usually, this is done for the pcramplified dna after round 7 or later selection rounds, depending on the enrichment of the binders. The topo ta cloning method combines the advantages of ta cloning with the ligation activity of topoisomerase i. Topo ta cloning kit from invitrogen biocompare product. Set up the following reaction in a microcentrifuge tube on ice. Topo cloning is a restriction enzyme and ligase free cloning method. Invitrogen topo ta cloning kit for sequencing, without. We would like to inform you about our new vectors for sequence and ligation independent cloning slic, an advanced version of the ligation independent cloning method lic. Pipet 2 l of each ligation reaction directly into the vial of competent cells and mix by stirring gently with the pipette tip. The linearized vectors supplied in ta cloning kits have a complimentary 3.
Ta cloning exploits the terminal transferase activity of some dna polymerases such as taq polymerase. Pcr cloning with lowno background a 500 bp pcr product incubated with the linearized vector in a 3. However, a lot of users still stick with the original topo ta cloning kit with the pcr2. Was wondering if it could be due to the ratio of ta vector. Ligating the dna to yield a plasmid containing the geneofinterest. The ta cloning method takes advantage of the terminal transferase activity of some dna polymerases such as taq polymerase. The cloning kits are available with a variety of competent cells available separately or without competent cells, depending upon your needs and budget. After pcr using taq polymerase, the fragments are cloned into plasmids by ta cloning taq amplified for sequencing. Pcr products are amplified using a taq dna polymerase which adds a single deoxyadenosine to the 3 end of the product. Using the classic tacloning kit from invitrogen to isolate individual pcr products from a heterogeneous amplification.
Cloning protocol for the geneofinterest into a plasmid. Before cloning a dna product, a pcr amplification strategy must be chosen. Molecular cloning strategiestraditional cloningta cloning. Using the classic tacloning kit from invitrogen to isolate. Protocols to transform chemically competent and electrocompetent e. This cloning technique utilizes the ability of thymine to hybridize to adenine in the presence of ligases. Learn how simple it is to get 95% recovery of your clone in just 5 minutes including how to set up the cloning. This dna ligation kit can also be used for ligation into tavectors. Its no wonder gateway cloning has been the goto choice for years, by researchers with varying experiencefrom beginners to advancedfor protein expression, functional analysis, and much more. Genetic engineering is used in thousands of laboratories around the world. This dna ligation kit can also be used for ligation into ta vectors. This video explains how invitrogen topo pcr cloning technology works. The ta cloning kits are shipped on dry ice and contain a box of ta cloning reagents box 1 and a box of one shot competent cells box 2. Ta cloning is one of the simplest and most efficient methods for the cloning of pcr products.
This lecture explains about ta cloning process in details. Cloning cloning methods ta and topo ta cloning embl. You get a sequence that appears to have a frame shift, causing two or more sequences to be overlaid in the chromatogram. Tvector cloning, or ta cloning, is a convenient method for cloning pcr products generated with taq dna polymerase. I performed a ta coning method using invitrogens topo ta kit using top10. In this method, vectors containing 5 thymine overhangs are used to accept pcr products in which additional 3 adenosine overhangs have been added on by the nature of taq polymerase ampli. The ta cloning strategy is both simple and much more efficient than bluntended ligation for the cloning of pcr products. This makes it possible to clone this pcr product directly into a linearized cloning vector with single, 3t overhangs. This allows direct ligation of pcr products in just 5 minutes.