Isolation of Recombinant Escherichia Coli Iptg Induced Taq Polymerase and Characterization Through Polymerase Chain Reactions%2c Western Blotting and Gel Electrophoresis

Isolation of Recombinant Taq Polymerase for PCR Isolation of Recombinant Escherichia coli IPTG induced Taq polymerase and characterization through polymerase chain reactions, Western Blotting and gel electrophoresis * Braeden Cowbrough1, Michael Atkins2, Christopher Bonner3 From the Faculty of Biochemistry Lab 3006 B Carleton University, Ottawa, ON K1S 5B6 *Running title: Isolation of Recombinant Taq Polymerase for PCR To whom correspondence should be addressed: Braeden Cowbrough, Faculty of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON, CAN, Tel: [phone]; Email: [e-mail] Keywords: PCR, Taq Polymerase, IPTG induction, salting out, thermostable polymerase Background: Taq polymerase is a thermostable enzyme essential in the PCR reaction that is isolated from recombinant E. coli. Results: Taq polymerase was isolated, the sample was not pure Taq, molecular weight found to be 114kDa. Conclusion: The methods isolated taq but an improvement of cation exchange and increase heat denaturation should be added. Significance: An improved method of Taq isolation offers a valuable resource for PCR. SUMMARY The aim of this experiment was to isolate recombinant Taq polymerase from E. coli bacteria induced by IPTG. This was done successfully through differential centrifugation, ammonium sulfate precipitation, and heat denaturation as confirmed by Western Blotting, rt-PCR and PCR reactions. The isolated protein sample was found to still contain remnants of host proteins as confirmed through SDS-PAGE with a Taq polymerase mass of 113.4 + 14.3 kDa. The activity of the isolated Taq was found to be 3922.3 + 192.9 bp per minute with a protein concentration determined through a Bio-Rad assay to be 1.88 + 0.11 mg/mL. The threshold of rt-PCR was found to be 20 cycles, with a melting temperature of 81°C confirming no primer dimer formation. A 1% agarose gel of PCR products revealed 5883.5 + 289.4 base pair lengths of double stranded

References: 10 Isolation of Recombinant Taq Polymerase for PCR TABLE 1 Taq Sample Dilution Factor 10 x Absorbance at 620 nm (+ 0.0005) 0.881 1.145 0.951 0.449 0.429 0.391 Protein Concentration (corrected) 4.10 + 0.47 mg/mL