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Ligases >
Quick Ligation™ Kit |
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Prices are in US dollars and valid only for US orders.
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- Fast - 5 minutes for cohesive or blunt ends
- Convenient - ligation performed at room temperature
- Flexible - suitable for all common ligation reactions
Description:
The Quick Ligation™ Kit enables ligation of cohesive end or blunt end DNA fragments in 5 minutes at room temperature (25°C).
2X Quick Ligation Reaction Buffer:
132 mM Tris-HCL
20 mM MgCl2
2 mM dithiothreitol
2 mM ATP
15% Polyethylene glycol (PEG 6000)
pH 7.6 @25°C
Ligation Time Course: LITMUS 28 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.
Applications:- Cloning into vectors
- Library construction
- TA cloning
- Linker ligation
- Recirculization of linear DNA
Kit Components:
Quick T4 DNA Ligase
Quick Ligation Reaction Buffer (2X)
Storage Conditions
Storage Temperature:
-20°C
Notes
Usage notes:- 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.
FAQs
- What factors can cause incomplete ligation and/or transformation using the Quick Ligation Kit?
- Why is my transformation not working and what control reactions should be run?
- What is the concentration of T4 DNA Ligase provided in the Quick Ligation Kit?
- Can conventional T4 DNA Ligase (400,000 units/ml) be used with the Quick Ligation buffer?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
The Quick Ligation Kit is tested for transformation efficiency using the following protocol. LITMUS 28 vector is cut with either EcoR V (blunt) or Hind III (cohesive), treated with calf intestinal phosphatase and gel purified. Blunt inserts from a Hae III digest of ΦX174 DNA and cohesive inserts from a Hind III of digest of λ DNA are ligated into the respective vectors at a 3:1 insert:vector ratio using the Quick Ligation Protocol. Ligation products are transformed as described. Each lot exceeds the following standards for efficiency (transformants/μg):
Recircularization: Cohesive ends (>5 x 106), blunt ends (>2 x 106), Uncut vector (2 x 107)
Insertion: Cohesive ends (>1 x 106), blunt ends (>1 x 106)
Companion Products
Quick Blunting™ and Quick Ligation™ Kits
Quick Blunting™ Kit
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