Other commercial preparations of t4 dna ligase may contain exonuclease activities that may remove the terminal deoxythymidines from the vector. The quick ligation kit enables ligation of cohesive end or blunt end dna fragments in 5 minutes at room temperature. Identification of the substancemixture and of the companyundertaking 1. In cloning, dephosphorylation prevents religation of linearized plasmid dna. Ligafast rapid dna ligation system promega corporation. The kit includes t4 dna ligase and a specially formulated 5x rapid ligation buffer optimized for fast and efficient dna ligation. Cip, bap or sap not completely inactivated from dephosphoryl. The convenient kit enables ligation of dna in 5 minutes and dephosphorylation in 10 minutes. It also joins dna fragments with either cohesive or blunt term. Recombinant t4 dna ligase is supplied in 25 mm trishcl ph 7. Partners in forensics, from lab, law enforcement, and public safety, can benefit from rapid dna results in many applications. The truseq dna library preparation kits are used to prepare dna libraries with insert sizes from 300500 bp for single, pairedend, and multiplexed sequencing. For blunt ends, use 1 l of t4 dna ligase in a 20 l reaction for 2 hours or 1 l high concentration t4 dna ligase for 10 minutes.
The rapid dna ligation kit contains all reagents necessary for ligation. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. Lexogen indexing solutions 6 nt indexing kits 12 nt unique dual indexing kits. The nextflex rapid xp dna seq kit is designed for 2.
The kit can be used to prepare single, pairedend, and multiplexed dna libraries for sequencing using illumina platforms. As a result a user can influence the outcome of their sequencing run by selecting the right kit. Thermo scientific rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 minutes at room temperature. Split rna extraction kit split rapid viral rna dna extraction kit. Lligation can be performed by using the rapid dna ligation kit or t4 dna ligase 0505. The applied bioystems rapidhit id system offers new ways to work together in the effort to solve more crime. Find and compare commercial and governmental sources for immunological and biological products using the linscotts directory search engine. Neb quick ligation kit, or neb bluntta ligase master mix.
Teloprime fulllength cdna amplification kit v2 lexogen. Linkeed rapid dna ligation kit quality control version 2. Nanopores sequence any strand of dna presented to them, independent of the length or source. The sparq dna library prep kit provides components for the rapid construction of dna libraries from fragmented doublestranded dna for sequencing on illumina ngs platforms. Adapter ligation thaw and prepare the kit reagents as follows. Sequencing library preparation for miniontm and promethiontm.
This kit contains the new recombinant rapid alkaline phosphatase, the tool of choice for dephosphorylation of 5 phosphates from nucleic acids. Rapid ligation buffer rlb supplied in this kit is specially designed for the efficient rapid ligation of dna. Classification 67548eec, 199945ec not a hazardous substance or mixture according to ecdirectives 67548eec or 199945ec. The rapid dna ligation kit enables stickyend or bluntend dna ligation in only 5 minutes at room temperature see fig. Dephosphorylation requires 10 min, while ligation of dna is performed in a 5 min reaction. This previously unreported activity may enable novel approaches for characterization of mirnas and mrnas, including snps. The following ligation reaction of a 3kb vector and a 0. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. The ends of dna fragments are joined together by the formation of. The ligateit rapid ligation kit includes usb t4 dna ligase, nucleasefree water, and a specially formulated 5x reaction buffer which enhances the activity of t4 dna ligase, especially on bluntend fragments. This kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into doublestranded plasmid dna. Fast ligation efficiency is equal to that obtained with t4 dna ligase in a standard 1 hour ligation. Weiss units reactions m8221 100 30 m8225 500 150 m8226 50 15 description.
Spikein rna controls sirvs spikein rna variant control mixes ngs data analysis. First, t4 dna ligase requires a 5 phosphate and a 3 hydroxyl group to. We do not recommend adding adaptor to a premix in the adaptor ligation step. The t4 dna ligase included in the kit enables direct ligation without extra purification steps before or after the ligation. Take forward approximately 700 ng of endprepped dna in 30 l into adapter ligation. What this product does number of tests the kit is designed for 40 or 160 dephosphorylation 1 g dna each and ligation reactions depending on pack size standard assay. A rapid and simple method for dna engineering using cycled. Dna recovery lanes 16 from hindiiidigested lambda dna lane c and lambda dna. For an unknown reason, while ligation efficiency appears to. Typical ligation reactions use 100200ng of vector dna. Previously, we developed a onestep sequence and ligationin dependent cloning slic method that is simple, fast, and costef fective. Ligafasttm rapid dna ligation system certificate of. Quantify 1 l of endprepped dna using a qubit fluorometer recovery aim 700 ng. The ligafast rapid dna ligation system is designed for the efficient ligation of stickyended dna inserts into plasmid vectors in just 5 minutes bluntended inserts in as little as 15 minutes.
X174 dna and cohesive inserts from a hindiii digest of. Assemble the following reaction in a sterile microcentrifuge tube. Rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 minutes at room temperature. Ligation is performed with 50 ng of vector using the neb quick ligation kit neb. Efficient adaptor ligation for the preparation of dsdna.
Amplificationfree, native dna sequencing and retained base modification amplificationbased for low dna amounts or quality ligation sequencing kit rapid field sequencing kit pcr sequencing kit rapid pcr sequencing kit. All of the components are in a readytouse format and do not require any additional reagents, such as atp. P c r y i e l d 100 n g ng 10ng 1ng pcr yields rapid. Use only the t4 dna ligase supplied with this system to perform pgem t and pgemt easy vector ligations. The kit contains t4 dna ligase and a speciallyformulated 5x rapid ligation buffer optimized for fast and efficient dna ligation. The lotus dna library prep kit can be used with a wide range of dna inputs 1250 ng and may be used in conjunction with idt adapters, including illumina truseq compatible custom adapters stubby or fulllength, xgen dual index umi adapterstech access, and ta ligation. The first step is an exponential amplification using standard primers and a master mix fomulation of q5 hot start highfidelity dna polymerase. Abb buffer abb at rt elution buffer elb at rt adapter mix 1d amx1d on ice running buffer with fuel mix. Splintr ligase, also known as pbcv1 dna ligase or chlorella virus dna ligase, efficiently catalyzes the ligation of adjacent, singlestranded dna splinted by a complementary rna strand. Dna extraction and library preparation for rapid genus and. The 5end dephosphorylation of vector is unnecessary for ligation reaction. Set up the following reaction in a microcentrifuge tube on ice.
As a result a user can influence the outcome of their sequencing run by selecting the right kit and protocol for their. Here we present a cycled ligationbased dna assembly protocol that is simple, cheap, efficient, and powerful. The wimp workflow can then be used to identify species from the resulting whole genome data, with the first results being generated in under 100 minutes from the start of the workflow. For blunt ends or single base overhangs, incubate at 16c overnight or room temperature for 2 hours alternatively, high concentration t4 dna ligase can be used in a 10 minute ligation.
Rapid dna lib prep kit produces the high rates of conversion to adaptorligated molecules. This previously unreported activity may enable novel approaches for characterization of mirnas and. Bbsihf or esp3i and ligation with t4 dna ligase into a single reaction, and can be used to. See appendix a for information on the design and the sequence of the marathon cdna adaptor. Dephosphorylation using quick dephosphorylation kit. Using the neb quick ligation kit heat inactivation of peg in the buffer ruins. Linkeed rapid dna ligation kit quality control version.
What this product does number of tests 40 ligation reactions standard assay. Ligation reactions performed using the 2x rapid ligation buffer do not need to be cleaned up before transformation. Dna extraction and library preparation for rapid genus. In molecular biology, ligation is the joining of two nucleic acid fragments through the action of an enzyme. Phusion sitedirected mutagenesis kit user guide pub. Ligation protocol with t4 dna ligase m0202 protocols.
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. Ligation products were transformed into chemically competent e. Thermo scientific rapid dna ligation kit for 150 reactions. The ligafast system is designed to eliminate any further purification prior to transformation of ligated 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 wimp workflow can then be used to identify species from the resulting whole genome data, with the first results being generated in under 100 minutes from the start of. Ligafast rapid dna ligation system protocolpdf 111 kb english. Following this reaction, adapters are added using the rapid genomic dna library kit, and the resulting library is loaded and sequenced. Litmus 28i vector was cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified. Locate proteins, assay kits, reagents, custom services. Takaras dna ligation kit, mighty mix, is a onesolution premix ligation reagent that offers high efficiency ligations, particularly for bluntended ligation.
Q5 sitedirected mutagenesis kit without competent cells. Alkaline phosphatase, calf intestinal ciap protocolpdf 111 kb english. Nebnext ultra ii dna library prep kit for illumina e7645. Ligafasttm rapid dna ligation kit promega can be effectively used in. The dna polishing reactions are combined in a single step to convert fragmented. Fast dna end repair kit t4 dna polymerase klenow fragment dntps clonejet pcr cloning kit rapid dna ligation kit t4 dna ligase standard cloning workflow fast cloning workflow dna fragment amplification, 1. T4 dna ligase and buffer combination for rapid 5minute ligations. The efficiency of the fast ligation is analogous to that obtained with t4 dna ligase in a standard 1 hour ligation. Store components at 20c in a non frostfree freezer. Switch to to see content relevant to your location. In a, the yields of fully ligated insert two adaptors are shown as a function of the molar excess of adaptor in the reaction 2. Step action 1 pipette the following into a sterile microcentrifuge tube on ice. Bluntended ligation of dna fragments purified using the zymoclean large fragment dna recovery kit.
Takaras dna ligation kit series are simple systems that allow very rapid dna. Rapid dna ligation kit t4 dna ligase standard cloning workflow fast cloning workflow dna fragment amplification, 1. Rapid dna kit was comparable to other companys kits. This kit will allow you to perform at least 8 reactions see page 4, warnings and precautions. This kit contains the new recombinant rapid alkaline phosphatase, the. Several gdna libraryprep protocols are available, offering low dna input, rapid reaction and ultralong reads fig. In order to test your ligase, try addingsome to dna ladder in ligase buffer, of course. Dna and rna sequencing kits oxford nanopore technologies.
Ligation buffer optimized for fast and efficient dna ligation. Rapid 1d and ligation sequencing kit 1d, allowing selection of the right tool for the right experiment. Incubate the reaction at room temperature for 5 minutes for cohesiveended ligations, or 15 minutes for bluntended ligations. The reaction can be performed for 5 minutes at 25c rapid protocol, or 30 minutes at 16c standard protocol with equal results for both circular and linear dna ligations. Fragments from plasmid dna digested with pvuii were purified, then mixed and ligated for the times indicated in the figure above. Dna ligation kit for fast and efficient dna dephosphorylation and ligation of sticky or bluntended. The protocol supports shearing by either sonication or nebulization with a low input requirement of 1 ug of dna. Ligateit kit rapid ligation rapid ligation of cohesiveend and bluntend fragments. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. The streamlined workflow can be completed in under 3 hours with minimal handson time. Set a 100 l or 200 l pipette to 80 l and then pipette the entire volume up and down at least 10 times to mix thoroughly. D4045, d4046 zymoclean large fragment dna recovery kit.
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