One of the most important steps in the cloning process is the ligation of linear DNA into a cloning vector. This ability to join fragments of DNA through recombinant technology is essential for many basic experiments in biotechnology. Examples include protein expression, mutagenesis, gene analysis and structure-function relationships. DNA ligations are performed by incubating DNA fragments with appropriately linearized cloning vectors in the presence of buffer, ATP, and DNA ligase.
The DNA ligation kit contains the reagents necessary to increase the consistency of ligations. This kit contains T4 DNA ligase, the enzyme of choice for virtually all cloning purposes because of its ability to ligate both cohesive and blunt-ended strands of DNA. The kit also contains Polyethylene Glycol (PEG) 8000, which enhances blunt-ended ligations by macromolecular crowding. A ligation control DNA is included as a system check. The DNA Ligation Kit is application-tested and contains no detectable DNase activity.
Many parameters affect ligations such as the relative ratio of insert to vector, the quality and type of the DNA ends, the temperature of ligation and the concentration of DNA. Each of these factors need to be considered for a successful ligation.
Vector DNA (0.033-1 µg/µL)
DNA fragment to be inserted (0.033-1 µg/µL)
Microcentrifuge tubes, 0.5 mL and 1.5 mL
A low temperature water bath may also be required
ATP = Adenosine 5’-triphosphate
PEG = Polyethylene glycol 8000
DTT = Dithiothreitol
EDTA = Ethylenediaminetetraacetic acid
The DNA Ligation Kit is for laboratory use only. Not for drug, household, or other uses. Kit contains components that are hazardous. See the MSDS before using.
Store at -20 °C