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DNA Polymerase I (5000 U)
Cat. No. / ID: P7050L
5000 U of DNA Polymerase I (10,000 U/mL) and 10x Blue Buffer. Storage temperature: –25°C to –15°C
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The DNA Polymerase I (5000 U) is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
The DNA Polymerase I is intended for molecular biology applications. This product is neither intended for the diagnosis, prevention or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.
Need bulk, customized or optimized products for commercial purposes? We also offer support with logistics, compliance and more. Reach out to cooperate with QIAGEN Strategic Partnerships & OEM
Features
- Synthesizes DNA in 5′-3′ direction
- Possesses both 3′→5′ and 5′→3′ exonuclease activities
- Enables nick-translation during DNA synthesis
Product Details
DNA Polymerase I is a mesophilic DNA polymerase that exhibits 5’-3’ DNA synthesis, in addition to both 3′→5′ and 5′→3′ exonuclease activities. The combination of DNA synthesis and 5′→3′ nuclease characteristics enable nick-translation during DNA synthesis.
The protein is produced by a recombinant E. coli strain carrying the PolA gene.
The enzyme is supplied in 25 mM Tris-HCl 0.1 mM EDTA 1.0 mM dithiothreitol 50% glycerol pH 7.4 at 25°C.
It is supplied with a 10X Blue Buffer (B0110) containing: 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM DTT, pH 7.9 at 25°C.
Performance
| Assay | Specification |
| Purity | >99% |
| Specific activity | 6850 U/mg |
| Single-stranded exonuclease | Functional |
| Double-stranded exonuclease | Functional |
| Double-stranded endonuclease | 200 U; no conversion |
| E. coli DNA contamination | 200 U; <10 copies |
Principle
DNA Polymerase I is a DNA-dependent DNA polymerase with inherent 3´→ 5´ and 5´→ 3´ exonuclease activities. The 5´→ 3´ exonuclease activity removes nucleotides ahead of the growing DNA chain, allowing nick-translation.
Procedure
Instructions for using DNA Polymerase I are provided in the corresponding kit protocol in the resources below.
Quality Control
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X Blue Reaction Buffer and added to 50 µL reactions containing Calf Thymus DNA, 1X Blue Reaction Buffer, 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 37°C, plunged on ice, and analyzed using a TCA-precipitation method.
Protein concentration was determined by OD280 absorbance.
Physical purity was evaluated by SDS-PAGE by loading a 2.0 μL of concentrated enzyme solution on a denaturing 4-20% Tris-Glycine gel flanked by a broad-range MW marker and 2.0 μL of a 1:100 dilution of the sample.
Single-stranded exonuclease was determined in a 50 μL reaction containing 15,000 cpm of a radiolabeled single-stranded DNA substrate and 10 μL of enzyme solution incubated for 4 hours at 37°C.
Double-stranded exonuclease activity was determined in a 50 μL reaction containing 15,000 cpm of a radiolabeled double-stranded DNA substrate and 10 μL of enzyme solution incubated for 4 hours at 37°C.
Double-stranded endonuclease activity was determined in a 50 μL reaction containing 1 μg of pBR322 DNA and 10 μL of enzyme solution incubated for 4 hours at 37°C.
E.coli contamination was evaluated using 5 μL samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using primers for the 16S rRNA locus.
Applications
This product is available for molecular biology applications such as:
- DNA labeling by nick translation
- Second-strand cDNA synthesis
Resources
Protocol Files (1)
Safety Data Sheets (1)
Certificates of Analysis (1)