Product Description
The Rapid Ligation Kit is a combination of T4 DNA Ligase and an optimized
reaction buffer designed to ligate blunt or cohesive end DNA fragments
in 5 minutes at room temperature.
Recommended Applications
Recommended applications include all routine
subcloning, recircularization of linear DNA, library construction, and
linker ligation.
Source of Protein
A recombinant E. coli strain carrying the cloned T4
DNA Ligase gene.
Components
1. T4 DNA Ligase supplied in 10
mM Tris-HCl, pH 7.4, 50 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, 50%
Glycerol.
2. 2X Rapid Ligation Buffer containing ATP.
Storage Conditions
Store at –20°C.
Unit Definition
One Weiss Unit is approximately equivalent to 67 Cohesive
End Ligation Units.
Quality Assurance
Purified free of contaminating endonucleases and exonucleases.
In addition, enzyme purity is analyzed by SDS-PAGE, and negligible E.coli genomic DNA is confirmed by qPCR.
Suggested Protocols
Rapid Ligation Protocol
1. To a sterile 1.5 mL tube,
combine 50 ng vector DNA with insert at a 3-fold molar excess.
2. Adjust
volume to 10 μL with sterile water.
3. Add 10 μL 2X Rapid Ligation
Buffer and mix.
4. Add 1.0 μL T4 DNA Ligase and mix gently.
5. Centrifuge
briefly to bring contents to base of tube, incubate 5 minutes at room
temperature.
6. Immediately transform 2 μL into competent cells. (Do
NOT heat inactivate.)
Basic Transformation Protocol
The following protocol is recommended
for transforming ligation products generated with the Rapid
Ligation Kit.
1. Thaw competent cells on ice.
2. Chill ~5 ng ligation mix
(2 μL) on ice in sterile microcentrifuge tube.
3. Add 50 μL thawed,
mixed competent cells to DNA and gently mix by pipetting.
4. Incubate
on ice for 30 minutes.
5. Heat shock at 42ºC for 2 minutes, immediately
return transformation mix to ice for 5 minutes.
6. Add SOC media (950 μL)
to cells, mix gently, and incubate at 37ºC for 1 hour.
7. Spread
100 μL of the reaction onto desired plate medium.
8. Incubate overnight
at 37ºC.
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