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T4 DNA Ligase Protocol

T4 DNA Ligase catalyzes the reaction of two cohesive- or blunt-ended strands of DNA, joining between the 5´-phosphate and the 3´-hydroxyl groups of adjacent nucleotides. The phage-encoded T4 DNA ligase is produced during an infection of E. coli by bacteriophage T4. T4 DNA ligase can ligate either cohesive or blunt ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. It can also ligate blunt-ended DNA with much greater efficiency than E. coli DNA ligase. The T4 DNA Ligase requires ATP as a cofactor.

GenieClone T4 DNA Ligase Product Components

Component MORV0108

10x Ligase Buffer

1ml

T4 DNA Ligase (400U/µl)

100µl


GenieClone T4 DNA Ligase Protocol

Component Amount

1.

Prepare the following reaction solution in a microcentrifuge tube:

Component Amount

10x Ligase Buffer

1 µl

Insert DNAa

0.3 pmol

Vector DNAb

0.03 pmol

T4 DNA Ligase (400 U/ µl)

1 µl

Sterile distilled water

10 µl
Note: 1. The molar ratio of Insert/Vector should be between 3: 1 and 10: 1.
            2. The blunt-end vector should firstly be dephosphorylated to avoid self-cycling.

2.

Incubate overnight at 16℃.

3.

Transformation.

3.1

Add the ligation product to 100μl of competent cells. The volume of the ligation product should be less than 1/6 of the volume of competent cells. Mix gently and incubate for 30 min on ice.

3.2

Incubate the mixture at 42℃ in a water bath for exactly 90 seconds. Then immediately chill on ice for 2 min-3 min without disturbing the mixture.

3.3

Add 900μl of LB or SOC medium to the centrifuge tube. Then out the tube in a shaker incubator (150rpm,37℃) for 45 min, during which the cells will recover and express the resistance gene.

3.4

Centrifuge at 2,500× g for 5 min and discard 900μl of supernatant. Resuspend the cells with the remaining medium and gently coated on a agar plate containing the appropriate antibiotics. Incubate overnight at 37℃.