T4 dna ligase thermo scientific

Thermo scientific t4 dna ligase product help molecular cloning. Dna insert ligation stickyend and bluntend into vector dna. Set up the following reaction in a microcentrifuge tube on ice. T4 dna ligase 5 ul thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. For convenience, ligations may be done at room temperature 2025c. The overall number of transformants may be increased. Thermo scientific rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 minutes at room temperature. The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. For cohesive sticky ends, incubate at 16c overnight or room temperature for 10 minutes. Salt t4 dna ligase will join cohesive end termini at salt concentrations as high as 300 mm without any loss in activity. Sinapse biotecnologia t4 dna ligase thermo scientific.

T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed. Ligation using t4 dna ligase amrita university youtube. One unit catalyzes the exchange of 1 nmol 32plabeled pyrophosphate into atp in 20 min. Thermo scientific t4 dna ligase, lc 1 ul t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Thermo scientific fastdigest enzymes product information. Winkel online voor een ruim aanbod thermo scientific t4 dnaligase t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed. T4 dna ligase is provided with 10x reaction buffer. Here we report a functional characterization of the t4 dna ligase. Ligation protocol with t4 dna ligase m0202 protocols. This section describes the general properties of the two enzymes. The two dna ligases share many properties, but also have some distinct properties that make them uniquely useful depending on application. The extracted dna can be further precipitated with ethanol. Together, these two sets of enzymes allow the recombination of two dna molecules in a predictable way. Thermo scientific t4 dna ligase, lc 1 ul fisher scientific.

The most commonly used is the t4 dna ligase method. Thermo scientific t4 dnaligase t4 dna ligase fisher scientific. Thermo scientific t4 dna ligase product help feb062015. Thermo scientific rapid dna ligation kit fisher scientific. After digestion, gel purification, and cuantification i performed a t4 dna ligase thermo scientific assay. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. This products resides on a fisher scientific gsa or va contract. T4 dna ligase catalyzes the formation of phosphodiester bonds between neighboring 3. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. Both blunt and cohesive end dna ligation, as well as singlestranded nick repair of dna, rna and dna rna, are. Singlestranded nucleic acids are not substrates for this enzyme. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. T4 dnaligase wird mit einem flaschchen mit 5x reaktionspuffer 250 mm trishcl ph 7. Singlestranded nucleic acids are not substrates for thi.

L t4 dna ligase 5 u water, nucleasefree total volume 20. The t4 dna ligase buffer should be thawed and resuspended at room temperature. Ligation buffer optimized for fast and efficient dna ligation. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english. The kit contains t4 dna ligase and a speciallyformulated 5x rapid ligation buffer optimized for fast and efficient dna l. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. What is the best temperature and incubation time for successful ligation. Greetings i have been struggling to ligate a 555 bp insert into a 6.

T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate. T4 dna ligase catalyzes the formation of a phosphodiester linkage between dna chains by condensing the 5phosphoryl group of one dna chain with the adjacent 3hydroxyl group of the other dna chain. Jun 01, 1997 atpdependent dna ligases are essential enzymes in both dna replication and dna repair processes. The overall number of transformants may be increased by extending the reaction time to 1 hour. T4 dna ligase from thermo fisher scientific selectscience. 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. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes.

One nterminal and two cterminal deletion mutants were expressed in escherichia coli as histidine tagged proteins. The enzyme used to ligate dna fragments is t4 dna ligase, which originates from the t4 bacteriophage. Thermo scientific t4 dna ligase product help molecular. In order to obtain the maximum amount of activity from the ligase, a ph of 7. T4 dna ligase is a ligation enzyme that can be used to join dna fragments by catalyzing the formation of phosphodiester bonds between juxtaposed 5 phosphate and 3 hydroxyl termini in doublestranded dna using atp as a coenzyme. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Applications cloning of restriction enzyme generated dna fragments cloning of pcr products joining of doublestranded oligonucleotide linkers or adaptors to dna sitedirected mutagenesis amplified fragment length. Active in themo scientific restriction enzyme, pcr, and rt buffers when supplemented with atp. Minimum passing specification is using t4 dna ligase amrita university. This chapter focuses on two of the best characterized dna ligases, the e. Learn more about how this product is being used in the product citation tool. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 moreminutes. What is the best temperature and incubation time for.

Linear vector dna 20100 ng insert dna over vector thermo scientific 10x t4 dna ligase buffer 2. Safely ligate samples with this buffer, for use with t4 dna ligase and tested for the absence of endo and exodeoxyribonucleases and ribonucleases. Although the reactions catalyzed by the enzymes of e. Singlestranded nicks in doublestranded dna are also closed by t4 dna ligase. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. There is considerable latitude in the temperature and time needed for successful ligations. Rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 minutes at room temperature. T4 dna ligase 200 units from thermo fisher scientific. The enzyme will not join singlestranded nucleic acids. Gently mix the reaction by pipetting up and down and microfuge briefly. Note the electrotransformation efficiency may be improved by. T4 dna ligase catalyzes the joining of two strands of dna between the 5phosphate and the 3hydroxyl groups of adjacent nucleotides in either a cohesiveended or bluntended configuration.

T4 dna ligase catalyzes the formation of a phosphodiester bond between the terminal 5 phosphate and a 3 hydroxyl groups of duplex dna or rna. It also joins dna fragments with either cohesive or blunt termini. T4 dna ligase rejoins dnas that have been cleaved by restriction enzymes. The enzyme repairs singlestrand nicks in duplex dna, rna, or dnarna hybrids. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. If you are viewing this page as a nonregistered user, the prices displayed is list price. Dna ligase is a specific type of enzyme, a ligase, ec 6. Sep 17, 2012 ligation using t4 dna ligase amrita university.

Active in thermo scientific restriction enzyme, rt, and t4 dna ligase buffers. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. Contents t4 dna ligase, supplied with 10x concentrated ligation buffer that includes atp. One unit is equal to approximately 300 cohesiveend ligation units. Fast ligation efficiency is equal to that obtained with t4 dna ligase in a standard 1 hour ligation. T4 dna ligase recombinant form of the enzyme from t4 phage. Thermo scientific t4 dna ligase is a single polypeptide.

Thermo scientific t4 dna ligase product help posted in molecular cloning. Prior to electrotransformation, remove t4 dna ligase from the ligation mixture by spin column or chloroform extraction. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Salt t4 dna ligase is insensitive to salt carried over from other reaction components vector or insert dna and allows ligation reactions to proceed in alternative reaction buffers with higher levels of salt e. It also joins dna fragments with either cohesive or blunt term.

T4 dna ligase catalyzes the formation of a phosphodiester bond between the terminal 5. For cohesive sticky ends, use 1 l of t4 dna ligase in a 20 l reaction for 10 minutes. The enzyme has also been shown to catalyze the joining of rna to either a dna or rna strand in a duplex molecule but will not join singlestranded nucleic. Thermo scientific t4 dna ligase 5 ul t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. L this protocol is for the dna insert ligation sticky end and bluntend into vector dna. The kit contains t4 dna ligase and a speciallyformulated 5x rapid ligation buffer optimized for fast and efficient dna ligation. T4 dna ligase buffer from thermo fisher scientific.