Ta cloning invitrogen protocol

The procedure exploits the terminal transferase activity of certain thermophilic dna polymerases, including thermus aquaticus taq polymerase. Pipet 2 l of each ligation reaction directly into the vial of competent cells and mix by stirring gently with the pipette tip. This enzyme adds a single, 3a overhang to each end of the pcr product. The pgemt vectors are a popular choice for general pcr cloning. 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.

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 yield of my purified pcr products is about 100 ng. Molecular cloning strategiestraditional cloningta cloning. 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. This post focuses on sticky end topo also called topo. 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 technique relies on the basic ability of complementary basepairs adenine a and thymine t to hybridize and form hydrogen bonds. Usually, this is done for the pcramplified dna after round 7 or later selection rounds, depending on the enrichment of the binders. Oct 12, 2011 however, each of these techniques has its own limitations. Invitrogen gateway cloning technology has been cited by life science researchers more than 2,000 times. This dna ligation kit can also be used for ligation into tavectors. Invitrogen topo ta cloning kit for sequencing, without.

Using the classic tacloning kit from invitrogen to isolate individual pcr products from a heterogeneous amplification. Centrifuge vials containing the ligation reactions briefly and place them on ice. However, a lot of users still stick with the original topo ta cloning kit with the pcr2. Ligating the dna to yield a plasmid containing the geneofinterest.

I performed a ta coning method using invitrogens topo ta kit using top10. Ta cloning is a simple and convenient method of subcloning polymerase chain reaction pcr products. 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. Given its importance it is remarkable that cloning strategies for many of the popular dna components are not standardised. This cloning technique utilizes the ability of thymine to hybridize to adenine in the presence of ligases. 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. Barber lab cloning with invitrogen topo ta cloning kit this procedure will insert your pcr fragment into a suitable vector in this case pcr2. Thaw, on ice, one 50 l vial of frozen one shot competent cells for each transformation. Topo ta cloning kit from invitrogen biocompare product. The pcr product is directly cloned into a ta cloning vector with a complementary 3t overhang in both ends without restriction digestion. 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.

This vector has antibiotic resistance ampicillian and kanamycin as well as having a gene for lacz production. 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 topo ta cloning method combines the advantages of ta cloning with the ligation activity of topoisomerase i. Using the classic tacloning kit from invitrogen to isolate. K202020 and k202040 are not supplied with one shot competent cells. Topo cloning is a restriction enzyme and ligase free cloning method. You should use this protocol in any the following circumstances. Pcr cloning with lowno background a 500 bp pcr product incubated with the linearized vector in a 3. Analyze agarose gels with longwave uv 360 nm to minimize uv exposure that may cause dna damage. This lecture explains about ta cloning process in details. After pcr using taq polymerase, the fragments are cloned into plasmids by ta cloning taq amplified for sequencing.

Protocols to transform chemically competent and electrocompetent e. May 27, 2015 this video explains how invitrogen topo pcr cloning technology works. The manual is clearly written and very easytofollow. Two protocols are provided to transform one shot chemically competent e. This dna ligation kit can also be used for ligation into ta vectors. 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 exploits the terminal transferase activity of some dna polymerases such as taq polymerase. In conclusion, this is a very useful kit for cloning pcr products which invitrogen has continued to improve since its release. The ta cloning method takes advantage of the terminal transferase activity of some dna polymerases such as taq polymerase. If screening of colonies containing plasmid with insert dna is necessary, we recommend doing. This makes it possible to clone this pcr product directly into a linearized cloning vector with single, 3t overhangs. Learn how simple it is to get 95% recovery of your clone in just 5 minutes including how to set up the cloning.

Set up the following reaction in a microcentrifuge tube on ice. Ta cloning kits for sequencing are shipped on dry ice. Dec 12, 2015 this lecture explains about ta cloning process in details. Consider the following factors when choosing the protocol that best suits your. The complementarity between the vector 3t overhangs and pcr product 3a overhangs allows direct ligation of taqamplified pcr products into the tvector, and this strategy is commonly referred to as ta cloning. 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. Barber lab cloning with invitrogen topo ta cloning kit.

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. The cloning kits are available with a variety of competent cells available separately or without competent cells, depending upon your needs and budget. Topo ta cloning kits are available from invitrogen. I performed the ta cloning and plated the cells at the same time. This video explains how invitrogen topo pcr cloning technology works. Ta cloning is one of the simplest and most efficient methods for the cloning of pcr products. No ligase, postpcr procedures, or pcr primers containing specific sequences are required. Kits containing competent cells include box with topo ta cloning reagents box 1 and a box with one shot competent. Invitrogens ta cloning kit dual promoter biocompare. Ta cloning is one of the simplest forms of cloning. I performed the ta cloning and plated the cells at the same time protocol.

Was wondering if it could be due to the ratio of ta vector. Pcr products are amplified using a taq dna polymerase which adds a single deoxyadenosine to the 3 end of the product. Invitrogen topo ta cloning kit for subcloning, without. Ligation protocol with t4 dna ligase m0202 protocols.

We have used the ta cloning kit extensively in our lab for cloning various genes and gene fragments. The ta cloning strategy is both simple and much more efficient than bluntended ligation for the cloning of pcr products. Cloning cloning methods ligation independent cloning lic. Ta cloning topo ta cloning provides a highly efficient, 5minute, onestep cloning strategy topo cloning for the direct insertion of. Cloning cloning methods ta and topo ta cloning embl.

First,i performed pcr reaction for 4 different genes using each gene specific primers. This allows direct ligation of pcr products in just 5 minutes. Perform pcr reaction as normal using pfu polymeraseif did pcr using taq skip to step 4. Quick protocol 1 pgemt and pgemt easy vector systems instructions for use of products a60, a80, a3600 and a3610. Before cloning a dna product, a pcr amplification strategy must be chosen. Genetic engineering is used in thousands of laboratories around the world. For example, ta cloning uses regular taq dna polymerase to add a single 3a overhang to the ends of the pcr product. The principle behind the ta cloning kit is quite simple. The dna ligation kit, mighty mix is a 2x ligation premix for highefficiency ligations, especially for bluntended ligations. I amplified my pcr products by using econotaq polymerase kit, then purify it using promega wizard purification kit. Here, a protocol using topo ta cloning kit invitrogen is shown. I put about 200 ng of pcr products into the ta cloning reaction with only 10 ng of ta vector. Topo ta cloning kit for sequencing supplied with the purelink quick plasmid miniprep cat.