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T4 DNA Ligase
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Molecular Biological Enzymes
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Product description
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Product Description
T4 DNA ligase catalyzes the formation of phosphodiester bonds between adjacent 5'-phosphate ends and 3'-hydroxyl ends on double-stranded DNA or RNA. The enzyme not only catalyzes the connection between blunt ends or sticky ends, but also repairs single-stranded cleavage in double-stranded DNA, RNA or DNA/RNA hybrid duplexes. It can be used to efficiently connect sticky and blunt ends, T/A cloning, dsDNA cutting and repair, constructing high-abundance clone libraries, and connecting RNA and DNA.
Specification: 600U/µL.
Transportation and storage methods
Ice pack transportation. Store the product at -20°C. The validity period is 2 years.
Activity definition
At 23°C, in 50 μL of 1×DNA ligase buffer, after incubating for 30 min, the amount of enzyme required to ligate 50% of 100 ng DNA fragments with sticky ends is defined as 1 activity unit (U). To
Quality Control
Exonuclease residue detection: In a 50 μL reaction containing a radiolabeled DNA substrate and 10 μL enzyme solution, incubate at 37°C for 4 hours, and the DNA electrophoresis band does not change.
Endonuclease residue detection: In a 50 μL reaction containing 0.5 μg plasmid DNA and 10 μL enzyme solution, incubate at 37°C for 4 hours, and the DNA electrophoresis band does not change.
E. coli Residual DNA Residual Detection: Use 5μL of denaturing enzyme solution to evaluate duplicate samples, and use oligonucleotide primers corresponding to the 16S rRNA locus to screen for the presence of contaminating E. coli genomic DNA in the TaqMan qPCR assay.
Operating procedures
1. Reaction system:
T4 DNA ligase catalyzes the formation of phosphodiester bonds between adjacent 5'-phosphate ends and 3'-hydroxyl ends on double-stranded DNA or RNA. The enzyme not only catalyzes the connection between blunt ends or sticky ends, but also repairs single-stranded cleavage in double-stranded DNA, RNA or DNA/RNA hybrid duplexes. It can be used to efficiently connect sticky and blunt ends, T/A cloning, dsDNA cutting and repair, constructing high-abundance clone libraries, and connecting RNA and DNA.
Specification: 600U/µL.
Transportation and storage methods
Ice pack transportation. Store the product at -20°C. The validity period is 2 years.
Activity definition
At 23°C, in 50 μL of 1×DNA ligase buffer, after incubating for 30 min, the amount of enzyme required to ligate 50% of 100 ng DNA fragments with sticky ends is defined as 1 activity unit (U). To
Quality Control
Exonuclease residue detection: In a 50 μL reaction containing a radiolabeled DNA substrate and 10 μL enzyme solution, incubate at 37°C for 4 hours, and the DNA electrophoresis band does not change.
Endonuclease residue detection: In a 50 μL reaction containing 0.5 μg plasmid DNA and 10 μL enzyme solution, incubate at 37°C for 4 hours, and the DNA electrophoresis band does not change.
E. coli Residual DNA Residual Detection: Use 5μL of denaturing enzyme solution to evaluate duplicate samples, and use oligonucleotide primers corresponding to the 16S rRNA locus to screen for the presence of contaminating E. coli genomic DNA in the TaqMan qPCR assay.
Operating procedures
1. Reaction system:
Reagent | 20 μL reaction system |
Carrier DNA | 20~100 ng |
Insert DNA | Molar ratio to carrier DNA 3:1 |
10× T4 DNA Ligase Buffer | 2 μL |
T4 DNA Ligase | 1μL |
ddH2O | Up to 20 μL |
2. Gently mix the reaction solution.
3. For sticky ends, incubate at 16°C overnight or at room temperature for 10 min; for blunt ends, incubate at 16°C overnight or at room temperature for 2 hours; high-concentration rapid ligase can be used for 10 min ligation reactions.
4. Transfer 1-5 μL of reaction solution to 50 μL of competent cells on ice.
Precautions
1. For your safety and health, please wear lab coats and disposable gloves for operation.
3. For sticky ends, incubate at 16°C overnight or at room temperature for 10 min; for blunt ends, incubate at 16°C overnight or at room temperature for 2 hours; high-concentration rapid ligase can be used for 10 min ligation reactions.
4. Transfer 1-5 μL of reaction solution to 50 μL of competent cells on ice.
Precautions
1. For your safety and health, please wear lab coats and disposable gloves for operation.
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