| Description
T4 DNA Ligase catalyzes the joining two strands of DNA between the 5' -phosphate and the 3' -hydroxyl groups of adjacent nucleotides in either a cohesive-ended or blunt-ended configuration(1). The enzyme has also been shown to catalyze the joinin of RNA to ither a DNA or RNA Strand in a duplex molecule but will not join single-stranded nucleic acids(1).
Features
Flexible: Use with 5' , 3' or blunt-dended DNA inserts.
Quality Control
1. Blue/White Cloning Assay: This assay is performed to demonstrate that T4 DNA Ligase is free from contaminating activities that can affect the efficiency and integrity of plasmid cloning. Any exonuclease or polymerase activity that alters the termini of linearized plasmids during ligation will result in a proportion of white-colored colonies above background levels.
2. Endonuclease Assay: To test for endonuclease activity, 1¦Ìg of Type I supercoiled plasmid DNA is incubated with 20 units of T4 DNA Ligase in 1X Ligase Buffer ( for 16 hours at 37 ¡æ . Following incubation, the supercoiled DNA is visualized on an ethidium bromide-strained agarose gel. There must be no visible nicking or cutting of the DNA.
3. Single-Stranded and Double-Stranded DNase Assay: To test for DNase activity, 50ng of radiolabeled single-stranded or double-stranded DNA is incubated with 20 units of T4 DNA Ligase in 1X Ligase Buffer for 16 hours at 37 ¡æ . Minimum passing specification is <2% release of single-stranded and <1% release of double-stranded radiolabled nucleotides as monitored by scintillaion counting of TCA-soluble material.
4. RNase Assay: To test for RNase activity, 50ng of radiolabeled RNA is incubated with 20 units of T4 DNA Ligase in 1X Ligase Buffer for 5 hours at 37 ¡æ . Minimum passing specification is <3% release of radiolabled nucleotides as monitored by scintillaion counting of TCA-soluble material.
5. Physical Purity: The purity is 3 90% as judged by SDS-polyacrylamide gels with Coomassie blue staining.
Applications
Joining double-stranded DNA molecules with cohesive or blunt ends. References
Engler, M.J. and Richardson , C.C. (1982) In: The Enzymes, Boyer, P.D., ed., Academic Press, New York , NY . Order Information |
