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Overexpression and FPLC‐purification of the DNA polymerase from T. aquaticus (779.7)
Author(s) -
Vidmar Kristen,
Parente Amy
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.779.7
Subject(s) - taq polymerase , thermus aquaticus , polymerase , dna polymerase , microbiology and biotechnology , polymerase chain reaction optimization , hot start pcr , biology , polymerase chain reaction , dna , inverse polymerase chain reaction , multiple displacement amplification , biochemistry , multiplex polymerase chain reaction , gene , dna extraction
Systematic evolution of ligands by exponential enrichment (SELEX) is a methodology in biochemistry for identifying nucleic acid sequences capable of adopting a shape that binds a target molecule. The process involves several rounds of the polymerase chain reaction (PCR) to amplify DNA sequences followed by steps that ultimately separate and enrich the population of DNAs with sequences having a high affinity for the target molecule. Four main reagents required for any PCR reaction include: 1 ‐ DNA template, 2 ‐ DNA primers, 3 ‐ dNTPs, and 4 ‐ DNA polymerase. PCR optimization is frequently performed to identify experimental conditions that are optimal for the chosen template and primers. This can be a costly step of the experimental procedures, as commercially available DNA polymerases can range from a few hundred to thousands of dollars. We have undertaken the process of overexpressing and purifying the heat‐stable DNA polymerase from Thermus aquaticus. A plasmid containing the gene for Taq DNA polymerase was transformed to BL21(DE3) E. coli cells. Cell cultures of these transformed cells can then be grown to saturation at 37°C and overexpression initiated with the addition of IPTG. The desired Taq polymerase will be purified from a portion of the cleared lysate using ion‐exchange chromatography. PCR activity using a standard template and primers can then be evaluated using samples of purified and unpurified Taq polymerase with comparison to several commercial samples.