Genescan 500 liztm size standard

Eight VNTR loci were selected and the forward primers were tagged with one of four fluorescent dyes, 6-FAM, VIC, NED or PET (Eurofins MWG/Operon). Primer pairs were combined in four duplexes, with the respective annealing temperature, as follows: XL-1 FAM and XL-4 VIC, 64°C; XL-13 NED and XL-15 PET, 60°C; XL-3 FAM and XL-5 PET, 64°C; XL-6 NED and XL-8 VIC, 68°C.
PCR products (1 μl of products marked with 6-FAM, VIC or NED; and 2 μl of products marked with PET) were diluted in ultrapure water to a final volume of 32 μl. An aliquot of 2.4 μl was mixed with 0.15 μl of the GeneScan^TM 500 LIZ^TM size standard (Applied Biosystems) and 9.35 μl formamide in a 96-well tray, followed by denaturation at 94°C for 10 min in a thermocycler. Capillary electrophoresis was conducted in the ABI3130 sequencer using the GeneMapper application. Chromatograms were visualized with PeakScanner^TM software v. 1.0 (Applied Biosystems). Fragment sizes were estimated and converted into copy number for each VNTR. To confirm the copy number for each VNTR locus, PCR products of strains CFBP 7660 and CFBP 7764 were sequenced at Genoscreen. Sequences were edited with GENEIOUS and the search tools were used to detect the tandem repeat sequences.

All C. glabrata isolates were typed with eight microsatellite markers (GLA2, GLA3, GLA4, GLA5, GLA6, GLA7, GLA8 and GLA9) as previously described by Brisse et al.37 (link). Genomic DNA was extracted using the NucliSENS^TM EasyMAG^TM (bioMérieux) system38 , eluted in 50 μl and stored at −20 °C. Amplification reactions were performed using a Lightcycler^TM 480 (Roche Diagnostics, GmbH, Germany) instrument with Lightcycler^TM 480 Probes Master (Roche Diagnostics, GmbH, Germany). The loci, primer sequences, fluorophores and hybridization temperatures are described in Table 4. The PCR products were visualized using 2% agarose gel electrophoresis in 1X of Tris borate EDTA buffer (Euromedex, France) with SYBR^TM safe DNA gel stain (Invitrogen, USA). Next, 1 μl of 1:100 diluted PCR products was mixed with a solution containing 25 μl HiDi formamide (Life Technologies, France) and 0.5 μl Gene Scan^TM 500 LIZ^TM size standard (Applied Biosystems, UK). The fragment length was determined via capillary electrophoresis using an ABI 3130 Genetic Analyzer (Applied Biosystems, France) and analyzed using GeneMapper software v4.0 (Applied Biosystems, France).

Prior to fragment analysis, PCR products from four different SSR primers were combined at 1:1:1:1 ratio. The multiplex (2.0 μl) was pretreated by adding 7.80 μl of Hi-DiTM Formamide (Applied Biosystems, USA) and 0.20 μl of GeneScanTM-500 LIZTM Size Standard (Applied Biosystems, UK) to make up 10.0 μl of the final volume. The mixture was vortexed thoroughly and followed by denaturation at 95 °C for 5 min. The plate was then placed on the ice immediately for another 5 min before capillary electrophoresis using ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, USA). The raw reads were retrieved and exported to GeneMapper V3.1 software. The genotype profiles were finally generated and displayed in the software for scoring purpose.