T4 DNA Ligase(Fast)

Storage and transportation conditions:-20℃

Product composition

注： 1 U=1 Weiss unit

Product introduction

T4 DNA Ligase(Fast) Carried by T4 bacteriophage Gene 30 of E. coli is produced. This enzyme catalyzes the double helix DNA or RNA between 5'- Phosphate groups and 3'- Phosphorus is formed between the hydroxyl groups Acid diester bond. The enzyme is double-stranded DNA、RNA or Repairable single strand between DNA/RNA complexes moment , And can be connected with sticky ends or flat ends DNA Fragments, but for single-stranded kernels Acid inactive, mainly used for restriction enzyme digestion products DNA Fragment cloning, gene localization change and PCR product cloning, linear DNA Self-cyclization and repair double strand DNA 缺刻。 T4 DNA Ligase(Fast) Need ATP was used as a cofactor to complete the viscous end joining reaction at room temperature in only 10 minutes.

Definition of enzyme activity unit

1 Weiss unit at 37 ° C The enzyme is present Catalytic conversion of 1 nmol [32PPi] to activated carbon adsorption state within 20 min. 1 Weiss unit Equivalent to about 200 viscous ends attached to reaction units (CEUs), equivalent to 30 min at 16 ° C inner connection 50% HindIII after digestion of lambda DNA fragment.

Enzyme activity detection conditions

Enzyme activity was tested in the following reaction mixture: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 66 μM ATP, 10 mM DTT, 3.3 μM [32PPi].

Quality Control

Detection of endonuclease residues

Combine 200 U of T4 DNA Ligase (Fast) with 1 μg of pUC19 DNA at 37 ° C medium incubation At 4 h, the change from covalently closed circular DNA to DNA with nicks was not detected.

Detection of exonuclease residues

Combine enzyme solution with double-stranded DNA The substrate is in The double-stranded DNA substrate was not changed by DNA electrophoresis.

Blue and white spot test

At room temperature, use 30 U T4 DNA Ligase (Fast) connect pUC57 DNA/ HindIII,pUC57 DNA/PstI pUC57 DNA/SmaI digestive product 1 h, then use E.coli XL1-Blue competent cells to transform the ligation products, and detectMeasured less than 1% white spots.

How to use

1. DNA The insert is attached to the vector DNA  (Adhesive end connection)

① Prepare the following reaction system on ice:

② Fully mixed and instantaneously separated, incubated at 22 ° C for 10 min;

③ take 1 ~ 5 μl The connection products are used for Transformation of 50 μl chemically competent cells, or take 1-2 μl for Transformation of competent cells by 50 μl electrotransformation.

NOTE: If ligation reaction products are used for electroconversion, a centrifuge column or chloroform extraction should be used to clean DNA instead of the thermal inactivation step.

2. DNA The insert is attached to the vector DNA  (Flat end connection)

① Prepare the following reaction system on ice:

② Fully mixed and instantaneously separated, incubated at 22 ° C for 1 h;

③ take 1 ~ 5 μl The connection products are used for Transformation of 50 μl chemically competent cells, or take 1-2 μl for Transformation of competent cells by 50 μl electrotransformation.

NOTE: If ligation reaction products are used for electroconversion, a centrifuge column or chloroform extraction should be used to clean DNA instead of the thermal inactivation step.