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T4 DNA Ligase can catalyze the bonding of sticky or blunt-ended double-stranded DNA or RNA between the 5'-P end and the 3'-OH end with a phosphodiester bond. At the same time, the enzyme can also repair single-stranded nicks in double-stranded DNA, RNA, and DNA/RNA hybrid chains.
Activity definition: One unit of enzyme can catalyze 1 nmol of 32P-labeled pyrophosphate into ATP through displacement reaction within 20 minutes at 37°C (one unit is equal to about 200 sticky-end attachment units).
T4 DNA Ligase Storage buffer：20 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 50% (v/v) glycerol。
5×T4 DNA Ligase Buffer：250 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 5 mM ATP, 5 mM DTT, Enhancer。
Storage and transportation
Transportation with wet ice; storage at -20°C, valid for 12 months.
T4 DNA Ligase
250 U (50 μL)
500 U (100 μL)
5×T4 DNA Ligase Buffer
Ligation reaction conditions
The ligation reaction at 25°C for sticky ends takes 5-30 minutes; the ligation reaction at 25°C for blunt ends does not exceed 2 hours or the reaction at 4°C overnight.
Conversion of ligation products
1. Take out 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 move the bottom of the tube with your fingers to mix, and bath in ice for 30 minutes;
3. Then place the product in a 42℃ water bath to heat shock for 90 s. After it is over, place it on ice quickly for 2-5 min in an ice bath;
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 hour to resuscitate the bacteria (it can also be placed in a 37°C incubator. Place culture for 1 h);
5. According to the experimental requirements, draw 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 the 37°C incubator and cultivate overnight.
Identification of positive clones
The monoclonal colonies grown on the plate are picked for colony PCR identification, or after culture, the plasmid is extracted by restriction digestion or PCR identification, or the extracted plasmid is directly sequenced and analyzed for identification.
1. It is recommended to configure the reaction system on ice.
2. When the concentration of the vector and the target fragment is low, it is not necessary to add water and directly use it to make up.
3. It is recommended that the molar ratio of vector DNA to inserted DNA fragment is 1:3~1:10.
4. A small amount of precipitation in the 5×T4 DNA Ligase Buffer is normal. It can be dissolved at 37°C before the experiment, and it will be used after mixing thoroughly. It has no effect on the experiment; it is recommended to dissolve in aliquots and freeze for use.
5. T4 DNA Ligase is recommended to be taken out when used and put back to -20℃ immediately after use.
6. When electroporation is used for transformation, the ligation product needs to be purified by column method or ethanol precipitation method.
7. When connecting the blunt-ended vector to the DNA fragment, the vector must be dephosphorylated (G3400 is recommended) to prevent its self-circularization.
8. Please wear lab coat and disposable gloves during operation.