Description
Endonuclease I hydrolyzes double- and single-stranded nucleic acids to oligonucleotides of 3-5 nucleotide lenghts with 5` – terminal phosphates.
Application: DNA and RNA degradation
Source: Isolated from Proteus vulgaris 84.
Unit definition: One unit is defined as the amount of enzyme required to cleave 1 μg of the indicated double-stranded oligonucleotide in 30 min at 37°C in a total reaction volume of 20 μl.
Application: DNA and RNA degradation
Assayed on:
Double-stranded oligonucleotide
5’-AGCAAATATTGCTCGATATC-3’
3’-TCGTTTATAACGAGCTATAG-5’
Reaction buffer: SE-buffer Endonuclease I
Storage conditions: 10 mM Tris-HCl (pH 7.4); 250 mM NaCl; 0,2 mM EDTA; 100 μg/ml BSA; 7 mM 2-mercaptoetanol ; 50% glycerol. Store at -20°C.
Quality control: 10 units of Endonuclease I is incubated with 20 ng of a single or double-stranded synthetic oligonucleotide in a 10 μl reaction mixture with the SE Endonuclease I Buffer for 3 hours at 37°C.Phosphatase contamination is determined by the loss of radioactivity from original 5` 32P-labeled oligonucleotide or products of 32P-labeled oligonucleotide digestion with Endonucleases I as determined by denaturating polyacrylamide gel electrophresis and subsequent autoradiography.
| Runs: 1. double-stranded synthetic oligonucleotide 5`p*GTGCATGGCGCCTATGCG3`/ 3`CACGTACCGCGGATACGC5` 2. double-stranded synthetic oligonucleotide 5`p*GTGCATGGCGCCTATGCG3`/ 3`CACGTACCGCGGATACGC5` plus Endonuclease I 3. single-stranded synthetic oligonucleotide 5`p*GTGCATGGCGCCTATGCG3`plus Endonuclease I 4. double-stranded synthetic oligonucleotide 5`p*GTGCATGGCGCCTATGCG3`/ 3`CACGTACCGCGGATACGC5` plus endonucleases Fai I (recognition sequence YA^TR) 5. single-stranded synthetic oligonucleotide 5`p*GTGCATGGCGCCTATGCG3` 6. single-stranded synthetic oligonucleotide 5`p*GTGCATGGCGCCTATGCG3`plus Alkaline Phosphatase labeled strand is marked by a symbol <*> |
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