| DNA Polymerase I Large Fragment exhibits the 5’ ---> 3’ polymerase and 3’ ---> 5’ exonuclease activity of DNA Polymerase. Lacks 5’ ---> 3’ exonuclease activity. |
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| Catalog # | D3940 |
| Applications | • Random primer labeling |
| • Sanger sequencing |
| • cDNA Second strand synthesis |
| • 3’ end-labeling, filling |
| Storage and Stability | Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. |
| CAS Number | 9012-90-2 |
| Molecular Weight | 62kD |
| Source | E.coli CM 5199 |
|
| Preparation | Prepared by subtilisin proteolysis of DNA Polymerase I, retaining the 5’ -3’ polymerase and the 3’-5’ exonuclease cayalytic activities of the native enzyme but lacks the 5’ -3’ exonuclease activity. |
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| Storage Buffer (Form) | Supplied as a liquid in 50mM potassium phosphate, pH 7.0 with 1mM DTT, 50% glycerol. |
|
| Concentration | ~10u/ul |
|
| Unit Definition | 1u catalyzes the incorporation of 10nmoles of deoxynucleotides into polynucleotide fraction using poly(dA-dT)•poly(dA-dT) as template•primer in 30 minutes at 37°C. |
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| Quality Control | |
| Activity Assay | |
| 67mM potassium phosphate, pH 7.4, 6.7mM magnesium chloride, 1mM 2-mercaptoethanol, 0.033mM dATP, 0.033mM dTTP, 0.4MBq/ml [H3]-dTTP, 62.5ug/ml poly(dA-dT)•poly(dA-dT). |
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| Endodeoxyribonuclease Assay | |
| No detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 20u of Klenow Fragment with 1ug of pBR322 DNA in 50ul of Reaction Buffer for 4hours at 37°C. |
|
| Function Assay | |
| Klenow Fragment was tested for filling-in recessed 3’-termini of DNA. |
|
| Exonuclease, Endonuclease, Ribonuclease | None detected |
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| Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
| Alternate names | E. coli lysogenic CM 5199 |
