The Quick Ligation Kit enables ligation of cohesive end or blunt end DNA fragments in 5 minutes at room temperature . (25°C )
For details on NEB's quality controls for DNA ligases, visit our Ligase Quality page.
Ligation Time Course:
LITMUS 28i vector was cut with either EcoRV (blunt) or HindIII (cohesive), treated with calf intestinal phosphatase and gel purified. Blunt inserts from a HaeIII digest of ΦX174 DNA and cohesive inserts from a HindIII digest of λ DNA were ligated into the respective vectors at a 3:1 insert:vector ratio using the Quick Ligation Kit. Ligation products were transformed into chemically competent E. coli DH-5α cells and grown overnight on LB-amp plates at 37°C.
1X Quick Ligation Reaction Buffer:
66 mM Tris-HCl
10 mM MgCl2
1 mM Dithiothreitol
1 mM ATP
7.5% Polyethylene glycol (PEG6000)
pH 7.6 @ 25°C
.
This product is related to the following categories:
Some of the most critical parameters which should be controlled to ensure successful ligation and transformation are addressed below. Cells: Competent cells can vary by several logs in their competence. Perceived ligation efficiency directly correlates to the competence of the cells used for transformation. Always transform uncut vector as a control for comparison purposes. Electroporation: Electroporation can increase transformation efficiency by several logs. Before using the products of a Quick Ligation reaction for electrotransformation, it is necessary to reduce the PEG concentration. We recommend a spin column purification. DNA: Purified DNA for ligations can be dissolved in dH2O (Milli-Q™ water or equivalent is preferable); TE or other dilute buffers also work well. For optimum ligation, the volume of DNA and insert should be 10 μl before adding 2X Quick Ligation Buffer. For DNA volumes greater than 10 μl, increase the volume of 2X Quick Ligation Buffer such that it remains 50% of the reaction and correspondingly increase the volume of ligase. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Insert:vector ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios. Time: Most ligations performed using the Quick Ligation™ Kit reach an end point at 5 minutes or less at 25°C. Incubation beyond this time provides no additional benefit. In fact, transformation efficiency starts to decrease after 1 hour and is reduced by up to 75% if the reaction is allowed to go overnight at 25°C. Biology: Some DNA structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies.
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
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The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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