Illustra autoseq g 50 dye terminator removal kit

B. microti positive products were purified using GFX PCR DNA and gel band purification kit (GE Healthcare Life Sciences, Buckinghamshire, UK) in accordance with the manufacturer's protocol. Purified samples were used for sequencing reactions carried out using a Big Dye Terminator v.3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA). The sequencing reaction product was purified using an Illustra Autoseq G-50 Dye Terminator Removal Kit (GE Healthcare Life Sciences, Buckinghamshire, UK) and 2 μL of sample was analyzed using an ABI3130 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA). Finally, the nucleotide sequences were analyzed by BLAST software and compared with sequences present in GenBank.

Genomic DNA was isolated from 0.5–1 g of frozen leaves using the “Illustra DNA extraction kit PHYTOPURE” (GE Healthcare) according to the manufacturer’s instructions. Then, genomic DNA was cleaned up using NucleoSpin Plant II (Macherey-Nagel). The DNA (20 μg) was digested with 40 U of HindIII in the presence of spermidine (5 mM) and BSA (0.1 mg/ml), then electrophoresed on a 0.8% agarose gel at 4.5 V cm⁻¹, and transferred on positively charged Hybond N⁺ membrane (GE Healthcare). DNA probe for the analysis, spanning a 341 bp-long portion of the 35 S promoter used to drive eGFP expression, was obtained by PCR with the following forward (F) and reverse (R) primers: F 5′-CTTCGTCAACATGGTGGAGCACGACA-3′ and R 5′-TGGAGATATCACATCAATCCACTTG-3′. The probe was labelled with dCTP [α ³²P] (Perkin Elmer) by random priming using “Prime-It II Random Primer Labeling Kit” (Stratagene). Unincorporated nucleotides were removed with the “Illustra AutoSeq G-50 Dye Terminator Removal Kit” (GE Healthcare). After overnight hybridisation at 42 °C in ULTRAhyb buffer (Ambion) in the presence of 10⁶ cpm ml⁻¹ of labelled probe, the blot was washed twice in 2X SSC containing 0.1% SDS at 42 °C for 5 min, and twice in 0.1XSSC containing 0.1% SDS at 42 °C for 15 min. Autoradiography was then performed using CL-XPosure film (Thermo Scientific).