T4 DNA POLYMERASE
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T4 DNA Polymerase Highlights
T4 DNA Polymerase is a mesophilic DNA polymerase that catalyzes 5' → 3' synthesis of DNA and has 3' → 5' exonuclease (proofreading) activity, but no 5' → 3' exonuclease activity. It removes 3' overhangs and fills-in 5' overhangs to form blunt ends.
- Efficiently blunt-end double-stranded DNA
- Reduce material costs with a drop-in solution that delivers equivalent performance at a differentiated price-point
- Custom formats, including high concentration, support lyophilization applications
- Highly stringent enzyme manufacturing ensures quality performance across lots
Applications
- Gap filling
- Generation of blunt DNA ends (removal of 3' overhangs and fill in 5' overhangs)
- Probe labeling via replacement synthesis
QC Specifications
| Description | Specification |
|---|---|
| Protein Purity Assay | ≥ 98% |
| Endonuclease Contamination Assay* | Not detectable |
| DNA contamination Assay ( E. coli , mammalian, library)* | < 10 copies |
| Phosphatase Contamination Assay* | < 1% released |
| Unit Assay* | Pass |
*As assessed using 2 U of enzyme input per assay.
Properties
Unit definition: One unit is defined as the amount of T4 DNA Polymerase that will incorporate 160 nmol of dNTPs into a DNA template in 60 minutes at 37°C
Reaction conditions:
10 mM Tris-HCl, pH 7.9
50 mM NaCl
10 mM MgCl₂
1 mM Dtt
Storage buffer: 1100 mM Potassium Phosphate, pH 6.5, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol
Heat inactivation: 75°C for 20 min
Molecular weight: 103.6 kDa
5' – 3' Exonuclease activity: No
3' – 5' Exonuclease activity: Yes
Strand Displacement activity: No
Fidelity: Moderate
Processivity: Moderate
BROCHURES
Precision Enzymes and Proteins Brochure
Custom Genomic Solutions Enzymes MDx DNA Polymerase I Equinox Library Amplification Equinox Uracil-Tolerant Library Amplification phi29 DNAP RNase Inhibitor StellarScript RT StellarScript HT RT StellarScript HT+ RT StellarTaq DNA Polymerase T4 DNA Ligase T4 DNAP T4 Polynucleotide Kinase T4 Gene 32 Protein Taq DNAPWatchmaker Genomics offers a portfolio of precision-engineered enzymes and proteins, designed to meet the demands of high-stringency applications. We provide tailored services such as custom fills, formats, packaging, and labeling to accommodate unique specifications.
PROTOCOLS
T4 DNA Polymerase Technical Guide
Enzymes T4 DNAPProtocol for T4 DNA Polymerase (3 U/µL) catalyzes 5'→3' DNA synthesis and has 3'→5' exonuclease activity, enabling gap filling and blunt-end formation for applications like library preparation.
| CONFIGURATION | |
|---|---|
| Kit contents | T4 DNA Polymerase (3 U/µL) |
| Unit definition | One unit is defined as the amount of T4 DNA Polymerase that will incorporate 160 nmol of dNTPs into a DNA template in 60 minutes at 37°C |
| Enzyme storage buffer | 100 mM Potassium Phosphate, pH 6.5, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol |
| QUALITY CONTROL | |
|---|---|
| Protein Purity Assay | ≥ 98% |
| Endonuclease Contamination Assay* | Not detectable |
| DNA contamination Assay* (E. coli, mammalian, library) | <10 copies |
| Phosphatase Contamination Assay* | <1% released |
| Unit Assay* | Pass |
*As assessed using 2 U of enzyme input per assay.
| SHIPPING AND HANDLING | |
|---|---|
| Shipping conditions | Ice packs |
| Storage | -20°C ± 5°C |
| Shelf life (off-the-shelf products) | ≥ 12 months |
| Shelf life (custom products) | ≥ 18 months |
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 of 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 for 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.
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| Description | 0.75 kU | |
|---|---|---|
| T4 DNA Polymerase (3 U/µL) | 7K0075-250UL | Request a quote |
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
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