T4 DNA Polymerase
Référence M005-01A
Conditionnement : 50U
Marque : Novoprotein
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Description T4 DNA Polymerase catalyzes the 5'→3' incorporation of nucleotides into double-stranded DNA using single-stranded and primed DNA as a template. It possesses strong 3'→5' exonuclease (proofreading) activity (100-1000 times stronger than that of Klenow fragment). Unlike E. coli DNA Polymerase I, T4 DNA Polymerase does not have a 5'→3' exonuclease function. Typical T4 DNA polymerase protocols include 5' DNA overhang blunting as well as the generation of blunt double-stranded DNA from double-stranded DNA containing 3' overhangs. These DNA products are often used in blunt cloning.
Usage - Blunting of DNA ends: fill-in of 5'-overhangs or/and removal of 3'-overhangs.
- Blunting of PCR products with 3'-dA overhangs.
- Probe labeling using replacement synthesis.
- Second strand synthesis in site-directed mutagenesis.
- Ligation-independent cloning of PCR products.
Quality Control After multiple column purifications, only a clear single target band was visible in SDS-PAGE gel detection; qPCR method detection showed no residual E. coli DNA and no endonuclease or exonuclease contamination.
Storage -20℃±5℃.
Components | Component | M005-01A (Size:50U) | M005-01B (Size:50U×5) |
|---|---|---|
| T4 DNA Polymerase (5U/μl) | 10μl | 10μl×5 |
| 10×T4 DNA Polymerase Buffer | 1ml | 1ml×5 |
Activity Definition One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C.
Notes - Heat inactivation: 70°C for 10 minutes.
- Keep the enzyme on ice when using it and store it at -20°C after use.
- The presence of Mg2+ is required for enzyme activity, and metal ion chelators can inhibit enzyme activity.
- When the ion concentration in the reaction system exceeds 100mM, the activity will be inhibited.
- Susceptibly affected by the higher-order structure of the template DNA, the T4 Gene 32 Protein can significantly increase the polymerase activity, but the 3'→5' exonuclease activity is completely inhibited.