What is T4 DNA Polymerase?
T4 DNA polymerase is a mesophilic DNA polymerase that catalyzes 5′ → 3′ synthesis of DNA. T4 DNA polymerase has 3′ → 5′ exonuclease (proofreading) activity but has no 5′ → 3′ exonuclease activity.
What is the unit definition of T4 DNA Polymerase?
One unit is defined as the amount of enzyme that will incorporate 160 nmol of dNTPs into a DNA template in 60 minutes at 37°C.
What are the applications for T4 DNA Polymerase?
- Gap filling
- Generation of blunt DNA ends (removal of 3′ overhangs or fill in 5′ overhangs)
- Probe labeling via replacement synthesis
Can T4 DNA Polymerase be used to remove 3’ overhangs and fill 5’ overhangs?
Yes, T4 DNA polymerase can both be used to remove 3′ overhangs and fill 5′ overhangs. T4 DNA polymerase is best used to blunt DNA at lower temperatures (12°C).
What is the storage buffer for T4 DNA Polymerase?
- 100 mM Potassium Phosphate, pH 6.5
- 1 mM DTT
- 0.1 mM EDTA
- 50% Glycerol
What buffer is recommended for T4 DNA Polymerase?
T4 DNA Polymerase performs well in a range of buffers. The recommended 10X buffer composition is a standard reaction buffer. This buffer should be optimized for specific applications.
Standard 10x T4 DNA Pol Reaction Buffer (not supplied with kit):
- 100 mM Tris-HCl pH 7.9
- 500 mM NaCl
- 100 mM MgCl₂
- 10 mM DTT
How can T4 DNA Polymerase be inactivated?
Heat inactivate through incubation at 75°C 20 minutes or through the addition of 2 µL of 0.5M EDTA per 50 µL reaction volume.
What is the optimal temperature for T4 DNA Polymerase activity?
To blunt DNA ends, we recommend all reactions be performed at 12°C.
What are the recommended reaction setup conditions?
Please reference our technical guide for instructions for use.
1. On ice, combine components as specified:
| Component | Final Concentration | Volume (50 µL reaction) |
|---|---|---|
| 10X Reaction Buffer | 1X | 5 µL |
| DNA | 0.5 – 2.0 µg DNA | Variable |
| dNTP | 100 μM of each | Variable |
| T4 DNA Polymerase (3 U/µL) |
1 unit per µg DNA | Variable |
| Water | Up to 50 µL |
2. Incubate as follows:
| Purpose | Temp | Time |
|---|---|---|
| Extension | 12°C | 15 minutes |
| Inactivation | 75°C | 20 minutes |
What is the extinction coefficient for T4 DNA Polymerase?
The extinction coefficient for T4 DNA Polymerase is 1.313 (mL/(mg*cm). If measuring protein concentration (A280), we recommend you use the protein’s extinction coefficient to ensure an accurate reading.
Do you offer custom formats?
Yes! Watchmaker offers customer fills, packaging, concentrations, and labeling – including private label – designed to meet your unique needs. We offer flexible terms to serve organizations of any size, and our right-sized processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com to learn more about our capabilities.
Are you ISO 13485-certified?
Yes! Our Quality Management System has achieved ISO 13485:2016 Certification. Our certificate has been awarded for the design, development, manufacture, contract manufacture, and support of high performing reagents for genomics applications in medical research. Download the certificate here.