诺唯赞试剂说明书 T4 DNA Ligase.docVIP

T4 DNA Ligase Catalog # : GM40501 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. This product is applicable to 3 end labeling of RNA, cyclization of RNA and DNA oligonucleotide and cDNA cloning. Components Components GM40501 40,000 U 10x Ligase Buffer 1 ml T4 DNA Ligase (400 U/μl) 100 μl Storage Conditions Stored at -20°C  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 (400 U / μl) 1 μl The molar ratio of the insert and vector should be among 3:1 to 10:1. For vector with blunt terminal, please perform the dephosphorylation of vector to prevent cyclization. Incubate the reaction mixture at 16°C overnight. Transformation Take the competent cells out of the -80°C refrigerator, and place the competent cells immediately in an ice water bath. Add the DNA into 100 μl of competent cells and mix gently. Keep in the ice for 30 minutes. Incubate the mixture at 42°C for 90 seconds. And then return to the ice bath for 2~3 minutes. Add SOC or LB medium up to a final volume of 1 ml. keep in water bath at 37°C for 10 minutes. Culture by shaking (150 rpm) at 37°C for 45 minutes to recovery. Centrifuged at 2500 g for 5 minutes, and then remove 900 μl of the supernatant. Mix the remaining solution and plate on selective media. Incubate at 37°C overnight. Unit Definition One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA in a total reaction volume of 20 μl in 30 minutes at 16°C in 1x T4 DNA Ligase Reaction Buffer. Protocol  Quality Control Exonuclease Activity: Incubation of 2000 U of this product and 0.6 μg of λ-Hind III at 74°C for 1 hour results in no detected change in DNA bands after gel electrophoretic. Endonuclease Activity: Incubation