DNA POLYMERASE I
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DNA Polymerase I Highlights
DNA Polymerase I is a mesophilic E. coli polymerase that catalyzes 5' → 3' template-directed DNA synthesis. DNA Polymerase I has 3' → 5' exonuclease (proofreading) activity and 5' → 3' exonuclease activity. It is capable of catalyzing de novo synthesis of synthetic homopolymers and provides a convenient method for the preparation of a variety of defined DNA substrates in the laboratory.
- Effective nick translation
- 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
- Nick translation
- Second-strand synthesis
QC Specifications
| Description | Specification |
|---|---|
| Protein Purity Assay | ≥ 99% |
| 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 DNA Polymerase I that will incorporate 300 nmol of dNTPs into a DNA template in 60 minutes at 37°C
Reaction conditions:
50 mM Tris-HCl, pH 7.9
10 mM MgCl₂
1 mM DTT
Storage buffer: 25 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 50% Glycerol, 1 mM DTT
Heat inactivation: 75°C for 20 min
Molecular weight: 103 kDa
5' – 3' Exonuclease activity: Yes
3' – 5' Exonuclease activity: Yes
Strand Displacement activity: No
Fidelity: High
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
DNA Polymerase I Technical Guide
Enzymes DNA Polymerase IProtocol for Watchmaker Genomics' DNA Polymerase I Kit which enables high-fidelity DNA synthesis, second-strand synthesis, and nick translation. Ideal for defined DNA substrates; store at -20°C.
| CONFIGURATION | |
|---|---|
| Kit contents | DNA Polymerase I (10 U/µL) |
| Unit definition | One unit is defined as the amount of DNA Polymerase I that will incorporate 300 nmol of dNTPs into a DNA template in 60 minutes at 37°C |
| Enzyme storage buffer | 25 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 50% Glycerol, 1 mM DTT |
| QUALITY CONTROL | |
|---|---|
| Protein Purity Assay | ≥ 99% |
| 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 DNA Polymerase I?
DNA Polymerase I is a mesophilic E. coli polymerase that catalyzes 5'→3' template-directed DNA synthesis.
What is the unit definition of DNA Polymerase I?
One unit of DNA Polymerase I is defined as the amount of enzyme that will incorporate 300 nmol of dNTPs into a DNA template in 60 minutes at 37°C.
What are the applications for DNA Polymerase I?
- Second-strand synthesis
- Nick translation
What is the storage buffer for DNA Polymerase I?
- 25 mM Tris-HCl, pH 7.4
- 0.1 mM EDTA
- 50% Glycerol
- 1 mM DTT
What is the recommended reaction buffer for DNA Polymerase I?
DNA Polymerase I 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.
Industry standard 10X DNA Polymerase I reaction buffer (not supplied with kit):
- 500 mM Tris-HCl, pH 7.9 at 25°C
- 100 mM MgCl₂
- 10 mM DTT
What are the recommended reaction setup conditions?
Please reference our technical guide for instructions for use.
- Depending on the application, add DNA Polymerase I at 0.25 – 1.0 U/μL and dNTPs at 25 – 100 μM final concentration in 1X DNA Polymerase I reaction buffer
- Incubate at 37°C for 30 minutes to 20 hours
- Stop the reaction by heating at 75°C for 20 minutes to inactivate the enzyme, or alternatively stop the reaction by adding an equal volume of 0.25M EDTA pH 8.0
Are reaction buffer and dNTPs supplied with the kit?
No, reaction buffer and dNTPs are not supplied with the kit.
What is the optimal temperature for DNA Polymerase I?
The optimal temperature for DNA Polymerase I activity is 37°C.
How is DNA Polymerase I inactivated?
DNA Polymerase I is inactivated by heating at 75°C for 20 minutes, or through the addition of an equal volume of 0.25M EDTA pH 8.0.
Can DNA Polymerase I be used to blunt DNA?
Yes, DNA Polymerase I can be used to generate blunt ends. Its 3'→5' activity can remove 3′ overhangs and the polymerase activity will fill in 5' overhangs.
What is the extinction coefficient for DNA Polymerase I?
The extinction coefficient for DNA Polymerase is 0.837. If measuring protein concentration (A280), we recommend you use the protein’s extinction coefficient to ensure an accurate reading.
Do you offer a drop in replacement for QIAGEN DNA Polymerase I?
Yes.
Product 7K0113-250UL is our drop-in replacement for NEB DNA Polymerase I.
Product 3K0084-250UL is our drop-in replacement for QIAGEN DNA Polymerase I.
For ordering, please Request a Quote or contact sales@watchmakergenomics.com.
Please reach out to support@watchmakergenomics.com for any other drop-in replacement inquiries.
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 | 2.5 kU | |
|---|---|---|
| DNA Polymerase I (10 U/µL) | 7K0113-250UL | Request a quote |
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
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