Abi 3730 sequencer

Polymorphisms in the pfmdr1 gene were determined by direct sequencing of the amplicons resulting from the nested PCR using each primer for target gene amplification. The sequencing was done using BigDye Terminator v3.1 cycle sequencing kit in an ABI 3730 sequencer (Applied Biosystems). The deduced amino acid sequences were aligned and analyzed with the Lasergene^® software (DNASTAR, Madison, WI, USA) using the reference sequences of 3D7 retrieved from Plasmodium database (http://www.plasmodb.org).

A subset of 109 strains collected from non-agricultural habitats were genotyped (Table 1). They were compared to 327 agricultural strains sampled from lettuce and tomato plants grown in several greenhouses in the South of France (Leyronas et al., 2015a ,b (link),c (link)).
Genomic DNA was extracted from aliquots of 15 mg lyophilized fungal material (harvested from two-week old cultures on Potato Dextrose Agar), following the DNeasy Plant extraction Kit protocole (Qiagen). The nine microsatellite markers designed for B. cinerea by Fournier et al. (2002) (link) were amplified following the protocol described by Leyronas et al. (2015b) (link). To determine the size of the microsatellites, the PCR products were scanned with the help of an ABI 3730 sequencer (Applied Biosystems). GeneMapper software version 4.1 (Applied Biosystems) was then used for the microsatellite size analysis. Complete microsatellite size profiles (referred to as “haplotypes” hereafter) were obtained for 109 environmental strains and 327 agricultural strains.

Screening for common ESBLs or carbapenemase genes, including bla_TEM (Yu et al., 2007 (link)), bla_SHV (Yu et al., 2007 (link)), bla_CTX-M (Yu et al., 2007 (link)), AmpC (Perez-Perez and Hanson, 2002 (link)), bla_KPC (Yigit et al., 2001 (link)), bla_IMP (Queenan and Bush, 2007 (link)), bla_{V IM} (Queenan and Bush, 2007 (link)), and bla_NDM (Zhang et al., 2013 (link)) were performed by PCR amplification using specific primers in a Tpersonal Cycler (Biometra, Germany). The PCR products were then sequenced using an ABI3730 Sequencer (Applied Biosystems, Foster City, CA, USA), and compared with the reported sequences from GenBank.

To investigate whether CFEM-containing proteins exist in plant7 (link) or not, we cultured tissues for Zea mays and Sorghum bicolor. Seeds were surface-sterilized by immersion in 75% alcohol for 1 min and in 0.01% mercuric chloride for 5 min, and then kept in 4 °C for 48 h. These seeds were dissected by peeling off the seed coat and endosperm. Acquired cotyledons were cultured on Murashige and Skoog (MSO) medium at 25 °C in an incubator for about two weeks under darkness until calluses were generated. All operations were carried out under sterile conditions in a laminar flow hood. Non-sterilized seeds were used as controls and cultured in the same conditions.
We used RT-PCR to amplify CFEM from total RNA and genomic DNA isolated from the cultured tissues of Z. mays and S. bicolor. For the first-strand cDNA synthesis, 1 μg of total RNA was reverse transcribed in a volume of 20 μl and stored at −80 °C for further use. Two pairs of primers (5′-GCTATTCCTTGCCTTGACGA CGCC-3′, 5′-CCGAGACCCTTGAGGCCAGCAGC-3′ for Z. mays; 5′-GGACGCTGGCGGAGCCTGTG-3′, 5′-TTGCCGCTCAGGACTTTGGTGG-3′ for S. bicolor) were designed from CFEM sequences identified from ESTs of the two plants. All products were isolated from a 1.5% agarose gel and cloned using the T-vector. Positive clones were cycle sequenced in both directions using Big Dye Terminator (Applied Biosystems, Foster City, CA) on an ABI3730 sequencer.

We performed Sanger sequencing for all the identified LGD_strict (likely gene-disrupting) mutations (including stop-gain, frameshift, and canonical splice-site variants) among the probands. DNA from parents and other family members was also examined to ascertain the inheritance pattern and segregation of the variant where applicable. Sanger sequencing was performed on genomic DNA from peripheral blood. Primers for Sanger sequencing (sequences on request) were designed with Primer 3 software and sequenced on an ABI 3730 sequencer (Applied Biosystems, Life Technologies). The data were analyzed with Sequencher 5.4.1 (Gene Codes Corp., Ann Arbor, MI).

PCR products (1–4 uL) were cloned into a pCR®-4TOPO vector using a TOPO TA Cloning® kit (Invitrogen) or pSTC1.3 vector (10 ul of PCR product) using a StabyCloning kit (Eurogentec, San Diego, CA). Plasmids from bacterial colonies cultured overnight in LB medium (100 ug/ml ampicillin) were purified using a QIAprep® Spin Miniprep kit (Qiagen). Plasmid inserts were sequenced using SequiTherm Excell (Epicentre Technologies, Madison, WI), as described [6] (link), or by the Colorado Cancer Center Core Sequencing Facility using an ABI3730 Sequencer (Applied Biosystems, Foster City, CA). Sequences are accessible in the GenBank database, www.ncbi.nlm.nih.gov/genbank.

Samples were tested using CBOL Plant Working Group^{9 (link)} recommended DNA regions rbcL, and matK as well as new primers designed in this study. The selected loci were amplified by polymerase chain reaction (PCR) on a PTC–100 thermocycler (Bio-Rad). DNA was amplified in 20 μL reaction mixtures containing 1 U AmpliTaq Gold Polymerase with GeneAmp 106 PCR buffer II (100 mm Tris–HCl pH 8.3, 500 mm KCl) and 2.5 mm MgCl2 (Applied Biosystems), 0.2 mm dNTPs, 0.1 mm of each primer (0.5 mm for matK), and 20 ng template DNA. Amplified products were sequenced for new markers in both directions with the primers used for amplification, following the protocols of the University of Guelph Genomics facility (www.uoguelph.ca/~genomics). Products from each specimen were cleaned using Sephadex columns and run on an ABI 3730 sequencer (Applied Biosystems). Bidirectional sequence reads were obtained for all the PCR products. Sequences were assembled using Sequencher 4.5 (Gene Codes Corp), and aligned manually using Bioedit version 7.0.9.