Sureclean pcr purification kit

Purified PCR products were ligated into the pCR4®-TOPO® TA cloning vector and transformed into One Shot® chemically competent Escherichia coli TOP10 cells (Invitrogen, Paisley, UK). Transformants were selected on Luria-Bertani (LB) agar plates containing ampicillin (50 μg ml⁻¹) and X-gal (80 μg ml⁻¹). Insert DNA from white colonies was amplified using the vector primers T7 (5′- TAA TAC GAC TCA CTA TAG G-3′) and T3 (5′- AAT TAA CCC TCA CTA AAG G −3′). Each PCR contained 1 μl of overnight culture, 1× PCR buffer, 1.5 mM of MgCl₂, 0.25 mM of each dNTP, 0.4 μM of each primer and 2.5 U of Taq polymerase (Bioline), and was made up to a final volume of 25 μl with sterile nuclease-free water (Ambion). The PCR was performed as described for the primers 27 F and 1492R, using 25 cycles. PCR products were purified using the SureClean PCR purification kit (Bioline). Sequencing analysis was performed using 0.5 μM of primer 27 F, a BigDye® Terminator v3.1 cycle sequencing kit and an ABI 3730 PRISM® Genetic Analyser (Applied Biosystems).

The genotypes showing different HRM profiles were sequenced using an ABI310 automatic sequencer and BigDye XTerminator Purification Kit (Applied Biosystems, Foster City, CA, USA). Specific primers (BD-SF: 5’-CCTACCCAGGTGTGCGGACC-3’; BD-SR: 5’-GTTTTCTTCATTGGGCCGAAC-3’) were designed to amplify an expected product size of 152 nucleotides and these fragments were bidirectionally sequenced to eliminate sequencing errors. PCR products were purified using SureClean PCR Purification Kit (Bioline, London, UK) following the protocol provided by the manufacturer.