It is purified and prepared from E. coli carrying a highly expressed T4 DNA Polymerase gene.
T4 DNA Polymerase, namely T4 DNA polymerase, is a template-dependent DNA polymerase, which can catalyze DNA synthesis reaction from the 5'→3' direction on a single-stranded DNA template bound with a primer. T4 DNA Polymerase has 3'→5' exonuclease activity, but no 5'→3' exonuclease activity, and its 3'→5' exonuclease activity is stronger than that of E. coli DNA polymerase I. It is mainly used in the blunting of 5' or 3' protruding ends of DNA, the synthesis of the second strand during site-directed mutagenesis, and the cloning of PCR products independent of ligation reaction, etc.
Component List
FP1508928 | Components | 300U | 3KU | 30KU | Storage |
FP1508928A | T4 DNA Polymerase (3U/μL) | 100 μL | 1.0 mL | 10 mL | -20℃ |
FP1508928B | 10 ×T4 DNA Polymerase Buffer | 1.0 mL | 10×1.0 mL | 100 mL | -20℃ |
FP1508928C | Solution I (100×) | 100 μL | 1.0 mL | 10 mL | -20℃ |
Scope of Application
1. Gap filling (no strand displacement activity) to generate blunt ends;
2. Filling 5' overhangs to form blunt ends;
3. Trimming 3' overhangs to form blunt ends;
4. Probe labeling via displacement synthesis;
5. Subcloning after single-strand deletion;
6. Synthesis of the second strand in site-directed mutagenesis.
Precautions
1. Since this enzyme has 3'→5' exonuclease activity, increasing the reaction temperature, increasing the enzyme amount, not adding dNTPs, or prolonging the reaction time may lead to the excision of bases at the DNA ends, resulting in recessed ends.
2. When using this enzyme, it should be stored in an ice box or on an ice bath, and immediately placed at -20°C for storage after use.
3. For your safety and health, please wear a lab coat and disposable gloves during operation.
Usage Method
Preparation before reaction system preparation:
a. Dissolve various reagents required for the reaction at 4°C, and place them on an ice bath or in an ice box for use. It is recommended to aliquot the reaction solution for use to avoid repeated freeze-thaw cycles.
b. The reaction system can be set up with reference to the end repair system in the table below, and it is recommended to prepare the system on an ice bath or in an ice box:
Component
| Volume
| Final Concentration
|
|---|
10×T4 DNA Polymerase Buffer | 5 μL | 1× | dNTP Mix* (25 mM) | 0.4 μL | 0.2 mM | Solution I (100×) | 0.5 μL | 1× | T4 DNA Polymerase (3 U/μl) | —** | — | Reactants
| —** | — | ddH₂O | To 50 μL | — | Final Volume
| 50 μL | — |
|
* 10×T4 DNA Polymerase Buffer does not contain dNTP Mix and needs to be prepared by customers themselves.
** It is recommended to add 1 U of T4 DNA Polymerase per microgram of DNA substrate.
Reaction Procedure (taking end repair as an example)
Step Name
| Temperature
| Time
|
|---|
Reaction
| 12~37℃ | 15~30 min | Inactivation
| 75℃ | 20 min | Storage
| 4℃ | ∞ |
|
Note: The reaction temperature and time can be adjusted according to the specific amount of substrate and enzyme.
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