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.
Figure 1: 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 nuclease-free water; 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. We recommend a vector amount of 20-30 fmol with an overall concentration of vector + insert between 1-10 ng/μl for efficient ligation. Concentration lower than 1 ng/μL may result in intramolecular ligation/circularization of the fragments. Vector: Insert molar ratios between 1:1 and 1:10 are optimal for single insertions (up to 1:20 for short adaptors). Use NEBioCalculator to calculate molar 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.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
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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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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![dsDNA substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] p OH [ straight line ] 5´](https://media.neb.com/m/71a7db236bf3c43a/Webp-LigaseTable_dsDNA_1.webp?=){kind=small}
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![dsDNA substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] p OH [ straight line ] 5´](https://media.neb.com/m/5f290e6356951d61/Webp-LigaseTable_dsDNA_4.webp?=){kind=small}
![Nicked substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] 5´](https://media.neb.com/m/463dbc5afc488218/Webp-LigaseTable_Nicks_8.webp?=){kind=small}
![Nicked substrate for ligase selection:5´ [ wavy line ] OH p [ straight line ] 3´3´ [ straight line ] 5´](https://media.neb.com/m/5662bda91f5de51d/Webp-LigaseTable_Nicks_4.webp?=){kind=small}
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