T5 Exonuclease (C007B-566415)

Aladdin's T5 Exonuclease is a specific exonuclease that degrades double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) in the 5' to 3' direction. It is able to initiate nucleotide removal from the 5' termini or at gaps and nicks of linear or circular dsDNA. However, it does not degrade supercoiled dsDNA. T5 Exonuclease also has ssDNA endonuclease activity, which can be inhibited by reducing the Mg2+ concentration to less than 1mM in the reaction mixture. T5 Exonuclease is widely used in Gibson Assembly.Gibson assembly is a simple and rapid approach for efficient assembly of DNA fragments with overlapped regions in a correct order. The reaction is carried out under isothermal conditions by three enzymesApplication：Gibson assembly; degradation of linear DNA and nicked plasmid DNA; removal of incomplete ligation products from ligated circular dsDNA; degradation of linear and nicked plasmid DNA to obtain high-purity supercoiled plasmid DNA; removal of the denatured DNA from alkaline-based plasmid purification methods; improve the transfection efficiency of mini extracted plasmid cDNA library.Source：Expressed and purified from E. coli transformed with the T5 phage D15 plasmid.Activity definition: One unit is defined as the amount of enzyme required to produce 1nmol of acid soluble deoxyribonucleotide from double-stranded DNA in 30 minutes at 37℃ in a total reaction volume of 50µl.Purity: Free from RNase, phosphatase, DNA endonuclease and other SNA exonucleases except T5 Exonuclease.Enzyme storage buffer：50mM Tris-HCl (pH7.5, 25℃), 100mM NaCl, 1mM DTT, 0.1mM EDTA, 0.1% (v/v) Triton X-100, 50% (v/v) glycerol.Inactivation or inhibition：T5 Exonuclease can be inactivated by adding EDTA to a final concentration of at least 11mM or by adding DNA loading buffer containing SDS to a final SDS concentration of 0.08%.Please see Figure 1 for the degradation of linear dsDNA, and Figure 2 for the degradation of linear ssDNA by the T5 Exonuclease from . Figure 1. Agarose gel analysis of linear dsDNA treated with T5 Exonuclease . One microgram of PCR products of 646bp in length were treated with different amounts of T5 Exonuclease from and Competitor, respectively, for 10 minutes at 37ºC in a total reaction volume of 50μl containing 50mM Potassium Acetate, 20mM Tris-acetate (pH7.9), 10mM Magnesium Acetate and 1mM DTT. The reactions were stopped by adding 1.2μl 0.5M EDTA and examined by 2% agarose gel electrophoresis.As shown in the figure,T5 Exonuclease has comparable performance in degrading dsDNA with the competitor product. Figure 2. Agarose gel analysis of linear ssDNA treated with T5 Exonuclease . One microgram of synthesized ssDNA of 59 nucleotides in length were treated with different amounts of T5 Exonuclease from and Competitor, respectively, for 30 minutes at 37ºC in a total reaction volume of 50μl containing 50mM Potassium Acetate, 20mM Tris-acetate (pH7.9), 10mM Magnesium Acetate and 1mM DTT. The reactions were stopped by adding 1.2μl 0.5M EDTA and examined by 2% agarose gel electrophoresis. As shown in the figure,T5 Exonuclease has comparable performance in degrading ssDNA with the competitor product.Precautions：T5 Exonuclease is a selective exonuclease that displays different activities to different DNA substrates. Therefore, the amount of enzyme and the incubation time should be optimized for different DNA substrates.The optimal reaction temperature of T5 Exonuclease is 37℃. But it is also active at 50ºC and therefore can be used for Gibson assembly.T5 Exonuclease is compatible with regular PCR buffers.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear lab coat and disposable gloves during the operation.Instructions for Use：1. Set up the reaction mixture on ice as follows:ReagentVolumeFinal DNAxµl0.02µg/µlNuclease-free Water(44-x)µl-10X Reaction buffer5µl1XT5 Exonuclease1µl0.2U/µlTotal Volume50µl-Note: T5 Exonuclease should be added at last, after mixing well all other contents. Keep T5 Exonuclease on ice during the reaction assembling.2. Mix the reaction well and have a quick spin to allow all the liquid accumulate at the bottom of the tube.3. Incubate at 37℃ for 10-30min.4. Immediately transfer the reaction on ice and terminate the reaction by adding EDTA to a final concentration of 11mM .5. Examine the DNA fragments by agarose gel electrophoresis or polyacrylamide gel electrophoresis. If DNA recovery from agarose gel is needed, use the DNA Gel Extraction Kit (, #D0056). Use the PCR Clean Up Kit (, #D0033) to purify DNA from the enzyme reaction mixture.

Specification: Free from RNase, phosphatase, DNA endonuclease and other SNA exonucleases except T5 Exonuclease.