T4 DNA Ligase

phosphodiester bonds between the 5′-phosphate terminal and 3′-hydroxyl terminal of the nucleic acid adjacent to the blunt or sticky end of dsDNA, and also catalyze the connection between RNA and ssDNA or RNA in DNA/RNA hybrids, but cannot catalyze the whole single-strand nucleotide connection. It is suitable for nucleic acid manipulation, such as labeling RNA 3′-terminal, cycling RNA and DNA oligonucleotides, and cloning cDNA.

torage and transportation

Store at -20 ℃, Transportation by dry ice；Valid for 2 years.

Unit Definition

One unit is defined as the amount of enzyme required to give 50% ligation of 6 μg Lambda-HindIII DNA in 30 minutes at 16°C in a total reaction volume of 20 µl.

Molecular Weight

55.3 kDa monomer.

Quality Control

Exonuclease Activity: Incubation of a 20 µL reaction containing a minimum of 2000 units of T4 DNA Ligase with 1 μg λ-Hind III digest DNA at 37℃ for 16 hours yields no degradation as determined by agarose gel electrophoresis.

Endonuclease Activity: Incubation of a 20 µL reaction containing a minimum of 2000 units of T4 DNA Ligase with 1 μg λDNA at 37℃ for 16 hours yields no degradation as determined by agarose gel electrophoresis.

Nicking Activity: Incubation of a 20 µL reaction containing a minimum of 2000 units of T4 DNA Ligase with 1 μg pBR322 at 37℃ for 16 hours results in <10% conversion to the nicked form as determined by agarose gel electrophoresis.

RNase Activity: Incubation of a 20 µL reaction containing a minimum of 2000 units of T4 DNA Ligase with 1.6 μg MS2 RNA for 16 h at 37 ℃ yields no degradation as determined by agarose gel electrophoresis.

E.coli DNA: 2000 units of T4 DNA Ligase is screened for the presence of E. coli genomic DNA using TaqMan qPCR with primers specific for the E. coli 16S rRNA locus. The E. coli genomic DNA contamination is ≤ 1 E. coli genome.

Reaction System and Condition

1. The ligation system is prepared in a microcentrifuge tube:

Note: In order to prevent self-ligation, the blunt end should be dephosphorylated before ligating. To improve the connection efficiency, 3 µL of 50% PEG 6000 can be added to the 20 µLreaction system.

2. Reaction condition: 16 ℃, overnight.
3. Transformation

1) Add the ligation product into 100 µL of competent cells (the volume of the ligation product should not exceed 1/10 of the volume of the competent cells), gently blend evenly, and incubate it on ice for 30 minutes.

2) Put the microcentrifuge tube in the 42 ℃ water without shaking. After accurate heat shock for 90 s, immediately put it on ice and let it stand for 2-5 minutes.

3) 900 µL LB or SOC medium is added into the tube, and the bacteria are revived after 45 minutes of shaking culture at 150 rpm and 37 ℃.

4) Centrifugation at 2,500×g for 5 minutes, remove 900 µL of supernatant and suspend the bacteria with the remaining supernatant. Gently smear the bacteria on a plate containing the correct resistance. When the bacteria liquid is absorbed by the plate, inverted culture overnight at 37℃.

Note: If super competent cells (Transformation efficiency > 108 CFU/µg) are used, 100-200 µL of a bacterial suspension at step 3) can be directly absorbed and coated on the plate. The remaining bacterial suspension can be stored at 4 ℃ and can be recoated within one week.

Usage Note

1. If a small amount of precipitation occurs during the melting of 10× Ligase Buffer, it is normal. Please mix it upside down before use.

2. Place T4 DNA Ligase on ice after taking it out of -the 20℃ refrigerator. Do not place it in the room above 25℃ for a long time.

1. Ligation of DNA fragment and vector DNA;

2. The connection between DNA fragment and Linker or Adaptor DNA.