The ends of the fragments are joined together by the formation of phosphordiester bonds between the 3 hydroxyl of one dna terminus with the 5phosphoryl of another. Dna ligation kit, version 1 and dna ligation kit, version 2. Using an optimized buffer system and t4 dna ligase, these dna ligation kits offer fast ligation in 30 minutes. This kit contains an optimized ligasebuffer system that enables ligation of long fragments without the need for extensive techniques or special expertise. Fast ligation efficiency is equal to that obtained with t4 dna ligase in a standard 1 hour ligation. Dephosphorylated dna with either blunt or sticky ends. Litmus 28 vector is cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified. The dna ligation kit contains the reagents necessary to perform both sticky and bluntend ligations. Pcr products were loaded on 1% agarose gels and extracted using a gel dna extraction kit ibi scientific. Assemble reaction mix into 10 l volume in a microfuge tube. L of ligation mix is sufficient for either chemical transformation or electroporation. Ligation sequencing kit 1d, ligation sequencing kit 2d or. As a result a user can influence the outcome of their sequencing run by selecting the right kit.
English canada download sdspdf 67 kb english united states. Rapid dna ligation kit 50 rxns from thermo fisher scientific supplier page view company product page. The kit has been optimized to the maximum efficiency and convenience in dna sample preparation workflow. Dna ligation kit, mighty mix a new single premix solution that offers quick, high efficiency ligation reactions, even for bluntended and tacloning reactions. Ligation reactions can be used directly in selected bacterial transformations with no need for dna.
Nanopores sequence any strand of dna presented to them, independent of the length or source. Property ligation sequencing kit 1d ligation sequencing kit 2d rapid sequencing kit sqklsk108 the simplest method for genomic dna, amplicon andor cdna with control over read length and amounts of input material sqklsk208 the highest accuracy for genomic dna, amplicon. Lk11025, lk0750h, and lk6201h connect with epicentre on our blog. Optional heatinactivate by incubating at 65c for 20 mins.
Cloning of fragments into either plasmid or phage vectors linker ligation recircularization of linear vector dna generation of libraries. The kit contains reagents for 150 ligations and is optimized for use with agilents lambda vector arms and plasmid vectors. Reaction may be scaled up to 20 l if dna concentrations are low. The specificity of ligation on the dna target is shown to allow the discrimination of a single point mutation with a drop in the ligation.
Thermo scientific rapid dna ligation kit fisher scientific. Assembly of dna parts into dna constructs is a foundational technology in the emerging field of synthetic biology. The kit contains t4 dna ligase and a specially formulated 5x rapid ligation buffer optimized for fast and efficient dna ligation. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation. X174 dna and cohesive inserts from a hindiii digest of. Dna ligase catalyzes the formation of two covalent phosphodiester bonds between the 3 hydroxyl group of one nucleotides and the 5 phosphate group of another in an atp dependent reaction. Rapid dna ligation kit california institute of technology. The mclabs dna ligation kit is used to ligate dna adapters to datailed dna fragments. I have compared the ligation efficiency of the rapid dna ligation kit to t4 dna. It is an essential laboratory procedure in the molecular cloning of dna whereby dna fragments are joined together to create recombinant dna molecules, such as when a foreign dna fragment is inserted into a plasmid. The kit contains t4 dna ligase and a speciallyformulated 5x rapid ligation buffer optimized for fast and efficient dna ligation. Linker ligation recircularization of linear dna kit components. T4 dna ligase catalyzes the formation of a phosphodiester bond between ends of adjacent, nicked nucleotides of dsdna, dsrna or dna rna hybrids.
Ligafast rapid dna ligation system protocolpdf promega. The efficiency of this fast ligation is analogous to that obtained with t4 dna. The rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 min at room temperature. The kit contains reagents for 150 ligations and is optimized for use with stratagene lambda vector arms and plasmid vectors. We have developed a novel approach called parallel analysis of rna ends pare for highthroughput identification of microrna mirna targets and diverse applications for the study of the. Property ligation sequencing kit 1d ligation sequencing kit 2d rapid sequencing kit sqklsk108 the simplest method for genomic dna, amplicon andor cdna with control over read length and amounts of input material sqklsk208 the highest accuracy for genomic dna. Before setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector to use for the ligation reaction. As a result a user can influence the outcome of their sequencing run by selecting the right kit and protocol for their. Standard t4 dna ligase can be heat inactivated at 65c for 20 minutes. Ligafasttm rapid dna ligation system product information. Aliquots of the recovered dna samples were ligated into pstibamhi double digested puc19 vector using the rapid dna ligation kit. Thermo scientific rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 minutes at room temperature.
Conveniently perform your entire cloning reaction in the same tube. Packaging 1 kit containing 5 components unit definition. English canada download sdspdf 66 kb francais france. Ligafast rapid dna ligation system protocolpdf 111 kb english. The kit contains t4 dna ligase and a speciallyformulated 5x rapid ligation buffer optimized for fast and efficient dna l. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. Enzymatic ligation methods are useful in the diagnostic detection of dna sequences. Protein detection using proximitydependent dna ligation. Do not heat inactivate the quick ligation kit or ligase master mixes.
Thermo scientific rapid dna ligation kit for 150 reactions. Fastlink dna ligation kit this product is exclusively available from lucigen corporation starting january 1, 2017. To perform a successful cloning experiment, use these handling steps as guidelines for using the rapid dna ligation kit. Agilent technologies dna ligation kit integrated sci. Some recombinant proteins are not well tolerated by e. Litmus 28i vector was cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified.
The table below provides information to guide the selection. The dna ligation kit includes t4 dna ligase for highly efficient ligations. Ligafast rapid dna ligation system promega corporation. The volume of vector dna and insert dna used in the ligation will vary depending on the size of each and their concentration. This kit contains the new recombinant rapid alkaline phosphatase, the tool of choice for dephosphorylation of 5.
The kit is optimized for use with our lambda arms and plasmid vectors. Here, we describe the investigation of nonenzymatic phosphorothioate. Ligation reactions can be used directly in selected bacterial transformations with no need for dna purification. The contents of the kit are ligation buffer, dna dilution buffer and t4 dna ligase. I have been using this kit for the past 2 years for cloning inserts of 24 kb into plasmid vectors. Quick ligation kit reach an end point at 5 minutes or less at 25c. Structural analysis of chromosome folding has been revolutionized by the chromosome conformation capture 3c family of techniques, which uses proximity ligation. Content and ordering information have been moved to. The rapid dna ligation kit can rapidly ligate dna with either blunt or sticky ends. Rapid 1d and ligation sequencing kit 1d, allowing selection of the right tool for the right experiment. Quick notes this kit provides a versatile, streamlined dna fragmentation and library construction protocol. The rapid dna ligation kit enables stickyend or bluntend dna ligation in only 5 minutes at room temperature see fig. The rapid dna ligation kit can rapi dly ligate dna with either blunt or sticky ends. Dephosphorylation requires 10 min, while ligation of dna is performed in a 5 min reaction.
Libraries for illumina sequencing may be prepared from a wide range of dna. Incubation beyond in fact, transformation efficiency starts to 75% if the reaction is allowed to go overnight some dna structures. The following protocol is recommended by new england biolabs. The ligafast rapid dna ligation system is designed for the efficient ligation of cohesiveended dna inserts into plasmid vectors in just 5 minutes bluntended inserts in as little as 15 minutes.
Biovision offers numerous dna ligation products in the molecular biology portfolio. Antibiotics, buffers, cloning, genetic, peptide synthesis, polymerase chain reaction, precipitation, purification, transformation. Rapid dna chemical ligation for amplification of rna and. The qiagen pcr cloning kit and box i of the qiagen pcr cloningplus kit containing ligation master mix and pdrive cloning vector should be stored at 20c in a constanttemperature freezer or at 70c immediately upon receipt. The superior function of mclabs dna ligation kit depends on our proprietary enzyme systems. Sep 25, 2007 the contents of the kit are ligation buffer, dna dilution buffer and t4 dna ligase. I have compared the ligation efficiency of the rapid dna ligation kit to t4 dna ligase from new england biolabs neb and mbi fermentas. Dna ligation is the act of joining together dna strands with covalent bonds with the aim of making new viable dna. The quick ligation kit enables ligation of cohesive end or blunt end dna fragments in 5 minutes at room temperature. Dna were ligated into the respective vectors at a 3.
Quick ligation products may be transformed by many different methods. The advent of in vitro dna amplification has enabled rapid acquisition of genomic information. Rapid dna, or rapid dna analysis, is a term used to describe the fully automated hands free process of developing a dna profile from a reference sample buccal cheek swab without human. Compare dna ligation kits from leading suppliers on biocompare. Box ii of the qiagen pcr cloningplus kit containing qiagen ez competent cells should be stored at 70c or. Mix approximately 30 pmol of acceptor piece with 10 pmol of isolated donor piece and 20 pmol of bridge dna oligonucleotide in. In molecular biology, ligation is the joining of two nucleic acid fragments through the action of an enzyme. Dna ligation is the act of joining together dna strands with covalent bonds with the aim of making new viable dna or plasmids. Chromatin packages the eukaryotic genome via a hierarchical series of folding steps ranging from individual nucleosomes to entire chromosome territories gibcus and dekker, 20. Ta cloning is a rapid method of cloning pcr products that utilizes stabilization of the singlebase extension produced by taq dna polymerase by the complementary t of the tvector prior to ligation.
Ligation products were transformed into chemically competent e. The quick ligation kit is tested for transformation efficiency using the following protocol. Construction of parallel analysis of rna ends pare. Our dna ligation kit for long fragments is a powerful tool for cloning long dna fragments spanning 2 kb to over 10 kb in length. Quick t4 dna ligase recombinant 2x quick ligation buffer quick t4 dna ligase supplied in. As a result a user can influence the outcome of their sequencing run by selecting the right kit and protocol for their experimental goals. Ligafasttm rapid dna ligation system certificate of. Incubation beyond in fact, transformation efficiency starts to 75% if the reaction is allowed to go overnight some dna structures, including inverted and tandem repeats, are selected e. Ligation sequencing kit 1d, ligation sequencing kit 2d or rapid sequencing kit. Mix approximately 30 pmol of acceptor piece with 10 pmol of isolated donor piece and 20 pmol of bridge dna. What this product does number of tests 40 ligation reactions standard assay.
We present here an analogous technique for protein detection, in which the coordinated and proximal. Plasmid cloning by pcr with protocols for ligation into a recipient plasmid. Dna ligation products nucleic acid kits molecular biology. T4 dna ligase and buffer combination for rapid 5minute ligations. This includes the 5 minutes dna ligation kit, t4 dna ligase, t4 rna ligases and e. These reagents are guaranteed to be dnase free while providing optimal ligation efficiency. Ligation sequencing kit 1d or rapid sequencing kit property ligation sequencing kit 1d rapid sequencing kit sqklsk108 the simplest method for genomic dna, amplicon andor cdna with control over read length and amounts of input material sqkrad002 rapid and easy method for genomic dna gdna, amplicon, cdna gdna 10 gb run 10 gb run input. Rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 minutes at room temperature. Depending on the dna concentration in the reaction, the ligation products will be either circular if the dna concentration is low or concatemeric if the dna concentration is high.
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