T4 DNA Ligase(5U/μl)
5×T4 DNA Ligation Buffer
In the presence of Mg2+ and ATP, T4 DNA Ligase catalyzes the formation of phosphodiester bonds between adjacent 5´ -phosphate and 3´ -hydroxyl ends on double-stranded DNA or RNA. This enzyme can not only catalyze the connection between the flat or sticky ends of double-stranded DNA, but also repair of double-stranded DNA, RNA or DNA/RNA hybridization double-stranded, but cannot catalyze the connection between single-stranded DNA.
Storage: Stored at -20℃ for one year.
Enzyme storage buffer:
10mM Tris-HCl (pH 7.5) ,50mM KCl,1mM DTT, 0.1mM EDTA,50% (v/v) glycerol .
5 × T4 DNA Ligase Buffer:
250mM Tris-HCl (pH 7.5), 50mM MgCl2,50mM DTT, 5mM ATP,Connection accelerator.
Source: Purified from E. coli carrying the T4 DNA Ligase gene.
Inhibitor and heat inactivation: NaCl or KCl greater than 200mM can strongly inhibit ligase activity. 65℃ heating for 10 minutes or 70℃ heating for 5 minutes can be inactivated.
The amount of enzyme required to exchange 32PPi of 1 nmol within 20 minutes at 37℃ is defined as a Weiss unit.The enzyme 1U(Weiss Unit) of this product corresponds to 200 viscous terminal units.In the T4 DNA ligase reaction buffer, the amount of enzyme required for 30 minutes to allow 50% of the lambda DNA fragment to be digested by Hind III at 16℃ is a cohesive terminal unit.
The results showed that the number of white spots was less than 2%. The excessive T4 DNA Ligase mixed superhelical plasmid assay showed no nuclease detection. The purity of recombinant protein was more than 99% by SDS-PAGE.
Product application: A connection between a sticky end and a flat end of double-stranded DNA; TA cloning; Repairing of double stranded DNA.
1.Connection reaction system
Add the following ingredients into a sterile 0.2ml PCR tube.
5×T4 DNA Ligase Buffer
T4 DNA Ligase (5U/μl)
Lightly flick the tube bottom with your finger and mix the ingredients. Centrifuge for a few seconds to make the reaction mixture sink to the tube bottom.
Sticky end connection: reaction at 25℃ for 5-10 minutes.
Flat end connection or one flat end connection with a sticky end connection: reaction at 25℃ for 30-60 minutes.
3.Usage of connecting vector and fragment
The amount of connecting vector and fragment is generally carried out in accordance with the molar ratio of 1:3-1:10. The usage of fragment is larger than that of vector. The usage of vector and fragment is generally between 50-100ng. For example, 3Kb vector and 0.5Kb fragment react at a molar ratio of 1:3. If a 50ng 3Kb vector is used, the segment requirement of 0.5Kb is calculated as follows:
After completion of the junction, 5-10μL of the junction products were added to the appropriate competent cells, and the transformation was carried out according to the transformation steps described in the instructions for competent cells.When the connection reaction is over and cannot be transformed, the product can be stored at -20℃.
1. After defreezing 5×T4 DNA Ligase Buffer, it should be mixed well before use.
2. The optimal activity temperature of T4 DNA Ligase is 37℃, but the conversion efficiency of the ligands formed by high-temperature Ligase will be greatly reduced, so the slightly lower connection temperature, such as 25℃ or 16℃, is selected. If the product does not need to be transformed and high connection effect is required, it can be carried out at 37℃.
3. The enzyme requires Mg2+ as the activator, So the presence of EDTA inhibits the connection reaction. DNA dissolved in a high concentration EDTA buffer needs to be replaced with sterile water or TE buffer.
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Beijing bomeide Gene Technology Co., Ltd. is a professional enterprise of DNA sequencing, DNA synthesis, molecular biological reagents and technical services.