诺唯赞试剂说明书 T4 DNA Ligase (Rapid)-N103-本人自己翻译的.docVIP

T4 DNA Ligase (Rapid) Catalog # N103 Introduction T4 DNA ligase catalyzes the formation of a phosphodiester bond between 5’ terminal phosphate and adjacent 3’ hydroxyl termini in duplex DNA, RNA or DNA/RNA hybrids. This enzyme will join blunt end and cohesive end termini but it cannot repair single stranded nicks. Package Information Component N103-01 600,000 U T4 DNA Ligase (Rapid) (600 U/μl) 1 mL 2 x Rapid Ligation Buffer 6 mL 10 x T4 Rapid Ligase Buffer 2 mL Reaction Buffer 2 x Rapid Ligation Buffer 132 mM Tris-HCI pH 7.6 @ 25℃ 20 mM MgCl2 2 mM DTT 2 mM ATP 15% PEG 6000 10 x T4 Rapid Ligase Buffer 500 mM Tris-HCI pH 7.6 @ 25℃ 100 mM MgCl2 50 mM DTT 10 mM ATP Storage Store at -20°C. Unit Definition One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (100ng)in a total reaction volume of 50 μl in 30 minutes at 23°C in 1x T4 DNA Ligase Reaction Buffer. Quality Control Exonuclease Activity: Incubation of 2000 U of this product and 0.6 μg of λ-Hind III at 37°C for 16 hour results in no detected change in DNA bands after gel electrophoretic. Endonuclease Activity: Incubation of 2000 U of this product and 0.6 μg of Supercoiled pBR322 DNA at 37°C for 4 hour results in no detected change in DNA bands after gel electrophoretic. Protocol Application example 1:Connect DNA and carrier 1. Prepare the ligation reaction mixture in a microcentrifuge tube: Sterile distilled water Up to 10 μl 10x Ligase Buffer 1 μl Insert a 0.3 pmol Vector b 0.3 pmol T4 DNA Ligase (Rapid) (600 U / μl) 1 μl a.The molar ratio of the insert and vector should be among 3:1 to 10:1. b.For vector with blunt terminal, please perform the dephosphorylation of vector to prevent cyclization. 2.Incubate the reaction mixture at 16°C overnight. 3.Transformation 1)Take the competent cells out of the -80°C refrigerator, and place the competent cells immediately in an ice water bath. 2)Add the DNA into 100 μl of competent cells and mix gently. Keep in th