Autoseq g 50 dye terminator removal kit

The 3′–5′ exonuclease activity of PfPolδ-cat was measured from the release of [α-³²P]dTMP from 3′ labelled poly(dA.dT) [16 (link)]. Substrate was prepared by incubating 125 μg/ml poly(dA.dT) with 5000 U/ml Klenow enzyme (New England Biolabs, MA, USA), 10 μM [α-³²P]dTTP in 50 mM potassium phosphate pH 7.5, 5 mM MgCl₂, 1 mM dAMP, and 0.5 mM β-mercaptoethanol. After incubation for 20 min at 37 °C, the reaction was termination by an addition of 10 mM EDTA and 1 M NaCl. The mixture was heated at 65 °C for 30 min and unincorporated [α-³²P]dTTP removed employing AutoSeq™ G-50 dye terminator removal kit (GE Healthcare). For detection of exonuclease activity, a 30-μl reaction mixture containing 50 mM HEPES pH 7.0, 40 μg/ml BSA, 10 % glycerol, 2 mM MgCl₂, 1.25 µg of 3′-labelled poly(dA.dT), and 0.2 μM PfPolδ-cat was incubated for 20 min at 37 °C and radioactivity measured as described above.

The PCR products were purified using a QIAquick Gel Extraction kit (Qiagen). The cycle sequencing reaction was following the protocol of the BrilliantDye Terminator v.3.1 kit (NimaGen, Nijmegen, The Netherlands; 1 µl BrilliantDye v 3.1, 3.5 µl 5× sequencing buffer, 1 µl template, 1 µl primer 5pMol, 13.5 µl water) using the PCR primers for sequencing. The cycle sequencing reactions were purified with an AutoSeq G-50 Dye Terminator Removal kit (GE Healthcare, Chicago, IL, USA). Sanger sequencing was carried out by capillary electrophoresis using a 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Obtained sequences were compared using the basic local alignment search tool (BLAST) provided by the National Center for Biotechnology (http://blast.ncbi.nlm.nih.gov/), with sequence records available in GenBank for confirmation of pathogen identification and species assignment.

Once the DNA was extracted, the specific sequences encoding the gene under study were amplified by PCR. Each amplification reaction was verified by electrophoresis through a 2% (P/V) agarose gel in Tris-acetate-EDTA (50×). Using Millipore Microcon columns (centrifugal filter device), the amplification products were separated from primers, proteins, salts, and resins. Direct sequencing of the aforementioned exons was performed using a Big-dye Terminator protocol according to the manufacturer’s instructions. Subsequently, each product was purified using Autoseq G-50 Dye Terminator Removal Kit (GE Healthcare, Little Chalfont, UK) columns and then processed by an automatic sequencer ABI PRISM (Applied Biosystems, Bedford, MA, USA).