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DC Field | Value | Language |
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dc.contributor.author | Maseh, Kashif | - |
dc.contributor.author | Syed Ali, Farhat | - |
dc.contributor.author | Ahmad, Shazeel | - |
dc.contributor.author | Rashid, Naeem | - |
dc.date.accessioned | 2024-04-25T10:48:29Z | - |
dc.date.available | 2024-04-25T10:48:29Z | - |
dc.date.issued | 2022-10-28 | - |
dc.identifier.citation | Maseh, Kashif, et al. "Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application." Preparative Biochemistry & Biotechnology 53.6 (2023): 704-711. | en_US |
dc.identifier.other | DOI | - |
dc.identifier.uri | http://10.12.5.105:8080/jspui/handle/123456789/1992 | - |
dc.description.abstract | Polymerase Chain Reaction (PCR) is widely used for cloning, genetic engineering, mutagenesis, detection and diagnosis. A thermostable DNA polymerase is required for PCR. Here we describe low-cost and high-recovery production of Pyrobaculum calidifontis DNA polymerase (Pca-Pol). The gene was cloned in pET-28a and expressed in Escherichia coli BL21CodonPlus. Gene expression conditions were optimized. Eventually, gene expression was induced with 0.1 mM IPTG for 3 hours at 37 C. Recombinant Pca-Pol produced was purified to homogeneity by immobilized metal-ion affinity chromatography yielding around 9000 U of Pca-Pol per liter of the culture with a recovery of 92%. Stability and PCR amplification efficiency of Pca-Pol was tested under various storage con ditions with highest efficiency in 25 mM Tris-Cl buffer (pH 8.5) containing 0.1% Tween 20, 0.2 mg/ mL BSA and 20% glycerol. Under this condition, no loss in PCR activity of Pca-Pol was observed, even after one year of storage. Repeated freeze-thaw, however, deteriorated enzyme activity of Pca-Pol. 55% PCR amplification activity retained after 7 prolong freeze-thaw cycles (freezing over night at 20 C and thawing for 45 minutes at 28 C). Purified Pca-Pol possessed 30 –50 exonuclease (proofreading) activity and is expected to have greater fidelity as compared to Taq polymerase which does not have proofreading activity | en_US |
dc.language.iso | en_US | en_US |
dc.publisher | Preparative Biochemistry & Biotechnology | en_US |
dc.title | Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application | en_US |
dc.type | Article | en_US |
Appears in Collections: | School of Life Sciences |
Files in This Item:
File | Description | Size | Format | |
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Pca cost effective 2022.pdf | 1.62 MB | Adobe PDF | View/Open |
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