Rapid DNA Dephos & Ligation Kit
ROCHE/RDLIG-RO - sufficient for 40 reactions (04898117001), sufficient for 160 reactions (04898125001), optimum reaction temp. 37 °C
Synonym: cloning of dna fragments; dna ligase; ligases
Product Type: Chemical
Site-directed mutagenesis by overlap extension PCR. Amplification of two different DNA polymerase mutant subfragments (Lanes 3 and 4), a short subfragment (Lane 1), and eGFP (Lane 2) to measure transfection efficiency. All fragments were cloned into the vector pIEX-1 (Novagen) for expression in the InsectDirect TM System (Novagen). After PCR amplification, fragments were cut out of the agarose and the DNA was purified with the High Pure PCR Product Purification Kit, followed by restriction enzyme digest in the respective SuRE/Cut Buffers (Roche) and ligation with the Rapid DNA Dephos and Ligation Kit.
Comparison of times required by classical cloning reagents and the Rapid DNA Dephos and Ligation Kit.
| manufacturer/tradename | Roche |
| parameter | 37 °C optimum reaction temp. |
| Quality Level | 100  |
| storage temp. | −20°C |
| usage | sufficient for 160 reactions (04898125001) |
| sufficient for 40 reactions (04898117001) |
| Application: | The Rapid DNA Dephos & Ligation Kit minimizes the time needed to perform diverse applications, including: • Cloning of fragments into either plasmid or phage vectors • Linker ligation • Recircularization of linear vector DNA • Generation of libraries |
| General description: | The Rapid DNA Dephos & Ligation Kit enables fast and efficient dephosphorylation and ligation of sticky- or blunt-end DNA fragments. |
| Other Notes: | For life science research only. Not for use in diagnostic procedures. |
| Packaging: | 1 kit containing 5 components |
| Specificity: | Specificity: rAPid Alkaline Phosphatase Alkaline Phosphatase catalyzes the hydrolysis of numerous phosphate esters, such as esters of primary and secondary alcohols, saccharides, cyclic alcohols, phenols and amines. Phosphodiesters do not react. The enzyme hydrolyzes inorganic pyrophosphate. The kinetic properties of the enzyme depend on many factors, such as purity of enzyme, concentration of enzyme in the assay, buffer, pH etc. Heat inactivation: rApid Alkaline Phosphatase: Two minutes at 75 °C. T4 DNA Ligase: 10 minutes at 65 °C. |
| Unit Definition: | Unit Definition: rAPid Alkaline Phosphatase One unit of rAPid Alkaline Phosphatase is the enzyme activity which hydrolyzes one μmol of 4-nitrophenyl phosphate in one minute at +37 °C under assay conditions. Volume Activity: rApid Alkaline Phosphatase: 1 U/μl T4 DNA Ligase: 5 U/μl |
| RIDADR | NONH for all modes of transport |
| WGK Germany | WGK 1 |
| Flash Point(F) | does not flash |
| Flash Point(C) | does not flash |
| Storage Temp. | −20°C |
| Enzyme Commission (EC) Number | 3.1.3.1 ( BRENDA  | IUBMB ) |
| UNSPSC | 12352200 |
| Components | rAPid Alkaline Phosphatase Buffer 10x concentrated; rAPid Alkaline Phosphatase 1 U/μl; DNA Dilution Buffer 5x concentrated; T4 DNA Ligation Buffer 2x concentrated; T4 DNA Ligase 5 U/μl |