T4 DNA Polymerase
Cat. No.: SB-G3456-150U
Size: 150U
Description
T4 DNA polymerase is a DNA polymerase derived from T4 bacteriophage, which is recombinantly expressed in Escherichia coli. It is a template-dependent DNA polymerase that can catalyze the 5'-3 'Directions for DNA Synthesis. It has 3'-5' exonuclease activity but no 5'-3' exonuclease activity (more active than DNA polymerase Klenow Fragment), and is a high-fidelity DNA polymerase. The 3'-5'DNA exonuclease activity of T4 DNA Polymerase is more active for single-stranded DNA than double-stranded DNA, that is, single-stranded DNA is easier to be digested by T4 DNA Polymerase than the unpaired strand part of double-stranded DNA . Based on these characteristics, it is mainly used for blunt-ended DNA 5' or 3' protruding ends, labeled DNA probe synthesis, site-directed mutagenesis, etc.
- Source : derived from the recombinant expression of T4 bacteriophage in Escherichia coli.
- Definition of enzyme activity: The amount of enzyme required to catalyze the incorporation of 10 nmol dNTP into polynucleotides within 30 min at 37°C is defined as 1 enzyme activity unit.
- Purity and concentration: SDS-PAGE detection purity > 95%; endogenous nucleic acid residue < 1 pg/μL (qPCR detection); 3 U/μL.
- Inactivation or inhibition: Incubate at 75°C for 20 min to fully inactivate; EDTA will inhibit activity.
- Enzyme Storage Buffer: 100 mM KPO4 , 1mM DTT, 50% glycerol, pH 6.5.
- 10x Reaction Buffer: 100mM Tris-HCl, 500mM NaCl, 100 mM MgCl2 , 1 mg/mL BSA, pH 7.9.
Storage and transportation
Transport in wet ice packs; store at -20°C, valid for 12 months.
Product Contents
| Component Number |
Component |
G3456-150U |
| G3456-1 |
T4 DNA polymerase |
50 μL |
| G3456-2 |
10× Reaction buffer |
500 μL |
| Product Manual |
1 copy |
Steps
blunt-ended DNA ends
1. Template and primer hybrid strand annealing : Mix the DNA template and primer equimolarly, the recommended final concentration is 10 μM, incubate at 90°C for 1 minute, and anneal through gradient cooling to 25°C to form hybrid double strands. 5' or 3' protruding terminal DNA.
2. Configure the reaction system according to the following table:
| Component |
Volume |
| 5' and/or 3' overhang dsDNA |
0.5 μL |
| 10х Reaction Buffer |
2 μL |
| dNTP Mix (2.5 mM each) |
1 μL |
| T4 DNA polymerase |
0.5 μL |
| Nuclease Free Water |
To 20 μL |
3. After configuring the reaction system, incubate at 12°C for 15 minutes or at 25°C for 5 minutes.
4. After the reaction is completed, heat at 75°C for 20 min to inactivate or add EDTA with a final concentration of 10 mM to terminate the reaction.
Precautions
- Since the enzyme has 3'-5' exonuclease activity, an increase in the reaction temperature, too much enzyme, no addition of dNTP or too long reaction time may cause the DNA 3' terminal base to be excised and form a concave end.
- All enzymes should be stored in an ice box and stored at -20°C immediately after use.
- For your safety and health, please wear a lab coat and disposable gloves for operation.
