KIT and PDGFRA mutation status in tumor samples was assessed by PCR using MyTaq HS DNA polymerase (Bioline, Germany) through 35 cycles with appropriate annealing temperatures. Primer sequences specific for KIT and PDGFRA mutations are shown in Supplementary Table 7 . PCR samples were then subjected to direct sequencing of single-stranded PCR products using BigDye® Terminator v1.1 cycle sequencing kit and the ABI Prism® 310 genetic analyzer (Applied Biosystems). For BRAF mutation analysis the cobas® 4800 BRAF V600 Mutation Test (Roche Diagnostics) was used according to the manufacturer's instructions.
Abi prism 310 genetic
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The ABI Prism® 310 Genetic Analyzer is a capillary electrophoresis-based system designed for DNA sequencing and fragment analysis. It features a single-capillary design and a laser-based detection system.
Automatically generated - may contain errors
Lab products found in correlation
Mytaq hs dna polymerase, by Meridian Bioscience (1 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Cobas 4800 braf v600 mutation test, by Roche (1 mentions)
Exosap, by Thermo Fisher Scientific (1 mentions)
Seqman software, by DNASTAR (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Pop 4 polymer, by Thermo Fisher Scientific (1 mentions)
5 protocols using abi prism 310 genetic
1
Genetic Mutation Analysis in Tumor Samples
2
Molecular Identification of Hepatozoon Species
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The species of Hepatozoon were identified through generating DNA sequences from PCR amplicons. For this, positive samples were purified using ExoSap (USB) and were sequenced in an automated sequencer (model ABI Prism 310 Genetic; Applied Biosystems / Perkin Elmer, California, USA), in accordance with the manufacturer's protocol, and with the same primers as used in the PCR. Sequences were trimmed for quality and edited by using the SeqMan software (Lasergene; DNAstar, Madison, Wis.). The partial sequences obtained were subjected to BLAST analysis (Altschul et al., 1990 (link)) to make inferences regarding the closest similarities to the sequences in GenBank.
3
16S rRNA Gene Fragment Analysis
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PCR products were purified using the QIAquick PCR Purification kit (Qiagen, Hilden, Germany). For each sample, 100 ng of the 16S amplicon were digested with 16 U MspI (New England Biolabs, Ipswich, replace with state) in a final volume of 5 µl at 37°C for 5 hours. The reaction was stopped by incubating the samples at 65°C for 20 min. The digested, fluorescently labelled fragments were detected using the ABI PRISM 310 genetic analyser (Applied Biosystems, Carlsbad, CA) with an injection time of 5 s and separation for 50 min at 15 kV in a Performance Optimized Polymer (Pop) 4 gel. A mixture of 1 µl of digested 16S amplicon, 9 µl of formamide, and 0.5 µl of GeneScan LIZ 1200 size standard (Applied Biosystems) was denatured at 95°C for 3 min and then placed immediately on ice. Each sample was analysed three times. The fragment lengths were determined with the GeneMapper ver. 4 software (Applied Biosystems) using the Local Southern Method.
4
Verification of emm Gene Presence by PCR
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The presence of the emm gene was verified by PCR as described by Podbielski et al. [8 (link), 13 (link)]. The PCR products were sequenced using an ABI Prism® 310 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany). The sequences obtained were compared with all available reference sequences on the United States (US) Center for Disease Control (CDC) website (http://www2a.cdc.gov/ncidod/biotech/strepblast.asp ). The emm gene was considered present if the degree of identity between the sequences reached 95 %.
5
Microsatellite Genotyping Protocol
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PCR with thirty-five cycles for 1 min each at 90 °C, 57 °C and 72 °C were performed. Amplified products (1 µl) were mixed with 12µl deionised formamide and 1 µl of 500 TAMRA Size standard (Applied Biosystems). Samples were denatured at 92 °C for 3min, then injected in ABI Prism 310 genetic analyzer (Applied Biosystems). Electrophoresis was performed in POP4polymer (Applied Biosystems), 15 Kv with 5 s injection time and 30 min electrophoresis time on a 47 cm 50 µm capillary (Applied Biosystems). After electrophoresis, fluorescence data was filtered and analysed using GeneScan analysis software version 3.1.2. Each haplotype is named according to the number of repeats at loci IVS8CA, IVS17bTA and IVS17bca, respectively.
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