Pcr2.1 topo vector

Identification of the T-DNA insert in Mu-Legacy was conducted using the PCR method [32 (link)]. DNA samples of nontransgenic ‘Legacy’ and Mu-Legacy were obtained using a cetyltrimethylammonium bromide (CTAB) method. Both HindIII-digested and EcoRI-digested DNA samples were ligated to adapters and then used for PCR using adapter primer AP1 and T-DNA border primers according to O’Malley et al. (2007) [32 (link)]. Eight primers to cover an approximately 200 bp region from each end of the T-DNA were designed based on the sequence of the T-DNA. Primer and adapter sequences used in this study are included in Additional file 7: Table S5. Target PCR products were recovered from gel, purified and ligated to a pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) for sequencing.
The identified 1279 bp sequence of the blueberry genome at the T-DNA insertion position was used to search the blueberry EST database (http://www.vaccinium.org). A 781 bp expressed sequence tag (EST) (CV091265) from nonacclimated floral buds, which has a 199 bp overlap with the 1279 bp sequence, was used as a reference to design the primers (i.e., E1F & E2R, E2F & E2R, and H2F & H2R) (Additional file 7: Table S5) for further extension of the 1279 bp sequence. PCR products were ligated to a pCR 2.1-TOPO vector (Invitrogen) for Sanger sequencing.

To verify HCV genotype and determine possible inter- and intra-patient subtype differences, HCV core amplicons (approximately 429 bp) were further analysed. A semi-nested PCR was performed with Sc2 and Ac2 as first-round primers and S7 and Ac2 as second-round primers [33 (link)] . The PCR conditions were as described for genotyping above. PCR products were purified from 2% agarose gel using QIAquick gel purification protocol (Qiagen Ltd., Germany) according to the manufacturer’s instructions. Purified amplicons were cloned directly into pCR 2.1-TOPO plasmid vector (~ 3.9 kb) and used to transform chemically competent Escherichia coli. Positive clones were detected through purification by Miniprep protocol (Qiagen Ltd.) and digestion with Eco RI. LB Agar, LB Broth Base, pCR 2.1 TOPO vector and Escherichia coli were obtained from Invitrogen, Life Technologies, Paisley, Scotland; and Eco RI was from Roche Diagnostics GmbH., Mannheim, Germany.
For each isolate, at least two clones were sequenced on both strands using BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). Sequencing products were purified by ethanol precipitation protocol. Electrophoresis and data acquisition were done on an automated ABI PRISM 310 genetic analyser (Applied Biosystems). Consensus nucleotide sequences obtained from the isolates were used in phylogenetic analysis.