4. 2 x Universal Ligation Mix ,$250

Product Information

Description

2x Universal Ligation Mix is a ready-to-use 2x master mix containing T4 DNA Ligase and reaction buffer. The contained T4 DNA Ligase catalyzes the binding of phosphodiester bonds between the 5′-P and 3′-OH ends of sticky-end or blunt-ended double-stranded DNA or RNA. At the same time, it can repair single-stranded nicks in double-stranded DNA, RNA, and DNA/RNA hybrid strands. The optimized premixed reaction buffer makes the reaction more efficient and easier to operate. The reaction system can transform a variety of chemically competent cells after 5 min at 25°C.

Storage and Handling Conditions

Transported with wet ice; stored at -20°C, valid for 12 months.

Component

Assay Protocol

Ligation system (recommended 10 μL reaction system)

Ligation reaction conditions

Sticky end ligation reaction at 25°C for 5-30 minutes; blunt end ligation reaction at 25°C does not exceed 2 hours or 4°C overnight.

Ligation product conversion

1. Take out the competent cells (such as E.coli DH5α, E.coli Top10, etc.) from the -80℃ refrigerator and place them on ice to thaw;

2. Add the reacted sample to the competent state, gently stir the bottom of the tube with fingers to mix, and place it in ice for 30 minutes;

3. The product was then placed in a 42°C water bath for heat shock for 90 s, and after that, it was quickly placed in ice for 2-5 min;

4. Take 900 μL of sterile SOC or LB medium and add it to the EP tube. After mixing, place the EP tube on a shaker and incubate at 220 rpm at 37°C for 1 h to recover the bacteria (it can also be cultured in a 37°C incubator for 1 h);

5. According to the experimental requirements, pipette different volumes of transformed competent cells and add them to the LB solid medium containing the corresponding antibiotics, spread the cells evenly, and after the liquid is completely absorbed, place the plate upside down in a 37°C incubator and cultivate overnight.

Positive clone identification

Pick the monoclonal colonies grown on the plate for colony PCR identification, or extract plasmids after culturing for digestion or PCR identification, or directly sequence and analyze the extracted plasmids for identification.

Note:

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

2. The vector and target fragments must be gel purified and their quality and concentration detected by electrophoresis. Water can be omitted when the concentration is low. When the total volume of vector and insert is greater than 5 μL, the reaction system can be scaled up to 20 μL.

3. The recommended molar ratio of vector to insert is 1:3~1:10.

4. When electroporation is used for transformation, the ligation product needs to be purified by column method or ethanol precipitation method.

5. It is recommended to take out the 2×Universal Ligation Mix before use, and put it back to -20℃ immediately after use. It can be frozen in aliquots to reduce repeated freeze-thaw cycles.

6. When the blunt-end vector is ligated with DNA fragments, the vector should be dephosphorylated (recommended G3400) to prevent its self-circularization.

7. Please wear lab coat and disposable gloves during operation.

For Research Use Only!

Ver. No.: V1.0-202111