2 x Universal T4 DNA Ligation Mix

Product Information：

2x Universal Ligation Mix is a ready-to-use mix containing T4 DNA Ligase and reaction buffer. The T4 DNA Ligase can be used to join DNA fragments with staggered or blunt ends and to repair nicks in double-stranded DNA having 3'-hydroxyl and 5'-phosphate ends. The mix are simple systems that allow very rapid and more efficient DNA ligation reactions.

Storage Conditions：

Ship with wet ice; store at -20°C, valid for 12 months.

Instructions for Use:

Perform ligation reaction

1. To an autoclaved, 1.5-ml microcentrifuge tube, add the following(recommend 10-uL reaction system):

2. Mix gently and centrifuge briefly to bring the contents to the bottom of the tube.

3. For cohesive ends, incubate at 25°C for 5-30 minutes; For blunt end, incubate at 25°C less than 2 hours or overnight at 4°C.

4. Place the tube on ice and proceed immediately to perform transformation reaction. Or you can store the ligation mixture at –20°C until you are ready.

Perform transformation reaction

5. Add appropriate ligation mixture into chemically competent cells (such as E.coli DH5α, E.coli Top10, etc.) and mix by tapping gently. Do not mix by pipetting up and down. The remaining ligation mixture(s) can be stored at −20°C.

6. Incubate for 30 minutes on ice.

7. Incubate for exactly 90 seconds in the 42°C water bath. Do not mix or shake.

8. Remove the centrifuge tubes from the 42°C bath and place them on ice for 2-5 minutes.

9. Add 900 µL of SOC or LB medium. Sterile technique must be practiced to avoid contamination. Shake the the centrifuge tube(s) at 37°C for 1 hour at 225 rpm in a shaking incubator.

10. Spread appropriate volume from each transformation centrifuge tube on separate, labeled LB agar plates. The remaining transformation mix may be stored at 4°C and plated out the next day, if desired.

11. Invert the plate(s) and incubate at 37°C overnight.

Analyze transformants

12. Select colonies and analyze by plasmid isolation, PCR, or sequencing.

Notice: 

1. It is recommended that the ligation reaction should be prepared on ice.

2. The vector DNA and insert DNA should be gel purified and analyse their quality and concentration by electrophoresis. Water can be omitted in ligation reaction if the concentration is low.

3. If the total volume of vector and insert is more than 5 μL, you may scale the ligation system to 20 μL.

4. For best results, 3:1-10:1 molar ratio of insert to vector is recommended.

5. If electroporation is used for transformation, vector DNA and insert DNA should be purified by column method or ethanol precipitation method.

6. The 2×Universal Ligation Mix should be kept at -20°C until within 5-10 minutes of use and returned

7. immediately to -20°C after use. It is recommended to freeze in aliquot to reduce freeze-thaw cycles.

8. If insert DNA is blunt end, the vector following restriction endonuclease digestion should be dephosphorylated (recommended G3400) to prevent its self-circularization.

9. For your safty and health,please wear safety glasses, gloves, or protective clothing.

10. For Research Use Only