9/13/2023 0 Comments Klenow fragment ligationThe Klenow fragment incorporates individual deoxyribonucleotides at a relatively modest rate of 50 nucleotides/s (6), and is moderately processive (on average, it synthesizes 50 nucleotides after binding and before dissociation). Much of the biochemical understanding of the polymerase and 3′-5 exonuclease functions stems from studies of the Klenow fragment. The N-terminal 5′-3′ exonuclease domain contains 323 amino acid residues, and the C-terminal fragment contains 605 residues (4, 5), which is frequently called the Klenow fragment. Pol I is cleaved into two subunits by mild proteolysis with trypsin. coli K12 chromosome (3), is a single-subunit enzyme that functions autonomously of other replicative factors. Subunits of Pol I and Their PropertiesĭNA polymerase I, a product of the PolA gene which maps at minute 86 of the E. The early cloning and overexpression of the Klenow fragment (1) and the determination of its three-dimensional structure (2) have allowed better understanding of polymerization mechanisms, molecular mechanisms of 3′-5′ exonuclease activity, and structure-function relationships of polymerases. coli (400 molecules per cell), and it functions primarily to fill DNA gaps during repair and replication. Pol I is the most abundant polymerase in E. coli DNA polymerase I (103 kDa) results in forming of two products: (1) the large Klenow fragment (68 kDa) that contains both the DNA polymerase and 3′-5′ exonuclease (proofreading) activities and (2) a smaller fragment (35 kDa) that contains the 5′-3′ exonuclease activity. This enzyme contains three activities: 5′-3′ polymerase, 3′-5′ exonuclease (for proofreading), and 5′-3′ exonuclease (for nick translation, excision repair, and hydrolysis of the RNA primers during DNA replication). Pol I, the first polymerase discovered in bacteria, is required for rapid, efficient growth in rich media. coli, has important roles during DNA replication, genetic recombination, and DNA repair. Pol I, one of three such polymerases found in E. Not for use in diagnostic procedures.Escherichia coli DNA polymerase I (pol I) has served for several decades as the prototype DNA-dependent DNA polymerase. Incubation of 10U of Klenow Fragment with supercoiled plasmid DNA produced no nicked molecules after 20 hours at 37 ☌ as determined by agarose gel electrophoresis analysis.įor research use only. Quality control: The enzyme is greater than 98% pure as indicated by SDS-polyacrylamide gel electrophoresis and contains no detected endonuclease activity. Note: When chewing back 3’ overhangs it’s important to include dNTPS as well, because in absence of dNTPs the 3’ →5’ exonuclease activity will lead to 3’ recessed ends. The Klenow enzyme is also active in Simple Restriction enzyme buffer and Taq Polymerase buffer when supplemented with dNTPs. Alternatively, the reaction can be stopped by adding EDTA to 10 mM final concentration. Add 1 unit Klenow per μg DNA and incubate 15 minutes at room temperature (25 ☌). Stop the reaction by heating to 75 ☌ for 20 minutes. Dissolve 0.1-4 μg of digested DNA in 1x Klenow reaction buffer supplemented with 40 μM each dNTP. Protocol for blunting DNA Fragments: Reaction conditions for removal 3’ overhangs and fill-in of 3’ recessed ends (5’ overhangs) are the same. Storage buffer: 0.1 M KPO 4 (pH 6.5), 1 mM DTT and 50% glycerol. Unit definition: One unit is defined as the amount of enzyme required to convert 10 nmol of dNTPs to an acid insoluble form in 30 minutes at 37 ☌.ġ0X Klenow Reaction Buffer: 500 mM Tris-HCl (pH 7.6 at 25 ☌), 50 mM MgCl 2, 10 mM DTTĪssay conditions: 50 mM Tris-HCl (pH 7.6 at 25 ☌), 5 mM MgCl 2, 1 mM DTT and dNTPs. Reagents supplied with: 10x Klenow reaction bufferĭescription: The Klenow Fragment lacks the 5’→3’ exonuclease activity of intact DNA Polymerase I but retains the 5’→3’ polymerase, the 3’ →5’ exonuclease and the strand displacement activities. coli strain carrying a plasmid with a clone copy of DNA Polymerase I large fragment Klenow Enzyme (DNA Polymerase I Large Fragment)
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |