The Applied Biosystems MD-10 linkage mapping panels (Applied Biosystems, Foster City, CA) were used to complete a genome-wide scan for family PKRP117. PCR was completed in a 5 μl reaction volume containing 40 ng of genomic DNA, various combinations of 10 μM fluorescently labeled primer pairs, 10X PCR buffer (100 mM Tris HCl pH 8.4, 400 mM NaCl, 15 mM MgCl2, 2.5 mM Spermidine), 2 mM deoxynucleotide triphosphate (dNTP) mix, and 0.2 U OneTaq DNA polymerase (New England BioLabs Inc, Ipswich, MA). Initial denaturation was performed for 5 minutes (min) at 95 °C, followed by 10 cycles consisting of denaturation at 94 °C for 15 s, annealing at 55 °C for 15 s and elongating at 72 °C for 30 s, and then 20 cycles consisting of denaturation at 89 °C for 15 s, annealing at 55 °C for 15 s, and elongation at 72 °C for 30 s. The final extension was performed for 10 min at 72 °C, followed by a final hold at 16 °C. PCR products were mixed with a loading cocktail containing HD-400 size standards (Applied Biosystems) and resolved in an Applied Biosystems 3100 DNA Analyzer. Genotypes were assigned using the Gene Mapper software from Applied Biosystems. Exclusion analyses were completed for PKRP262, PKRP344, and PKRP358 using closely spaced short tandem repeat (STR) markers.
3100 dna analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 3100 DNA Analyzer is a capillary electrophoresis-based system designed for DNA sequencing and fragment analysis. It utilizes fluorescence detection technology to analyze DNA samples. The instrument provides high-throughput and reliable data generation for various genetic analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Genemapper software, by Thermo Fisher Scientific (5 mentions)
Hd 400 size standards, by Thermo Fisher Scientific (5 mentions)
Md 10 linkage mapping panels, by Thermo Fisher Scientific (4 mentions)
Onetaq dna polymerase, by New England Biolabs (1 mentions)
Bigdye terminator kit, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Bigdyedeoxy terminators, by Thermo Fisher Scientific (1 mentions)
9 protocols using 3100 dna analyzer
1
Genome-wide Linkage Mapping for PKRP117
2
Genome-wide linkage analysis using STR markers
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The Applied Biosystems MD-10 linkage mapping panels (Applied Biosystems, Foster City, CA) were used to complete a genome-wide scan for family PKCC001. Multiplex polymerase chain reaction (PCR) was completed as described previously [23 (link)]. PCR products were mixed with a loading cocktail containing HD-400 size standards (Applied Biosystems) and resolved in an Applied Biosystems 3100 DNA Analyzer. Genotypes were assigned using the Gene Mapper software from the Applied Biosystems. Exclusion analysis was completed for PKCC113 using closely spaced STR markers. The sequences of the primer pairs used for exclusion analysis and amplification conditions are available upon request
3
Mutation Analysis of RB1 Gene
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The first phase of mutation analysis was performed on tumor DNA. The coding exons, flanking intronic regions, and the core promoter of the RB1 gene were amplified by PCR using commercial PCR kits and automated thermocycler (PE 2700; Applied Biosystems [ABI]). When the DNA was obtained from fresh tissue or leukocytes, we were able to amplify all 27 exons via primers designed using Primer3 software (http://fokker.wi.mit.edu/primer3/ ). For DNA extracted from paraffin-embedded tissue sections, new primers allowing for smaller amplicons (less than 150 bp) were designed. PCR products were purified enzymatically using ExoI/SAP for removal of excess primers and dNTPs and subjected to Sanger sequencing using one of the PCR primers. Sequencing reactions were performed on an Applied Biosystem 3100 DNA analyzer using Big Dye Terminator kit (Applied Biosystems). Sequences were compared to the reference sequence of the RB1 gene (GenBank L11910.1) using the Sequencher Aligner software (Sequencher 5.0. Gene Codes, Ann Arbor, MI, USA). Alterations of normal splice site were predicted by using bioinformatics tools (available at http://www.fruitfly.org/seq_tools/splice.html ). Prediction of the damaging effects of novel missense mutations was performed using PolyPhen 2 bioinformatics tools (available at http://genetics.bwh.harvard.edu/pph2/ ).
4
Silencing lncRNA16 in Lung Cancer Cells
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The lung cancer cell lines, PC9 and A549, were cultured at 37°C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, in a humidified incubator containing 5% CO2. The PC9 cell line was authenticated by DNA sequencing with 3100 DNA Analyzer (Applied Biosystems, USA). LncRNA16-shRNA (GenePharma, Shanghai, China) and empty vector, lncRNA16 and empty vector were transfected. The sequence for lncRNA16-shRNA were synthesized commercially, and were of sequences: 5′-GCCAGCGUU ACAGUAAUGUTT-3′ (sense primer); and 5′-ACAUUAC UGUAACGCUGGCTT-3′ (antisense primer). After 6 h, the transfection media was replaced with normal media, and transfected cells were cultured for another 24 h. The transfected cells were selected in medium containing puromycin.
5
Autosomal Recessive CSNB Genotyping
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Exclusion analyses were performed for reported regions of autosomal recessive CSNB with fully informative polymorphic short tandem repeat (STR) markers flanking the CSNB locus or gene. PCR products were mixed with a loading cocktail containing HD-400 size standards (Applied Biosystems, Foster City, CA) and resolved in an Applied Biosystems 3100 DNA Analyzer. Genotypes were assigned using the Gene Mapper software from Applied Biosystems.
6
Genome-wide Linkage Mapping for PKCC025
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Applied Biosystems MD-10 linkage mapping panels (Applied Biosystems, Foster City, California) were used to complete a genome-wide scan for family PKCC025. Multiplex polymerase chain reaction (PCR) was completed as described previously [29 (link)]. PCR products were mixed with a loading cocktail containing HD-400 size standards (Applied Biosystems) and resolved in an Applied Biosystems 3100 DNA Analyzer. Genotypes were assigned using the Gene Mapper software from Applied Biosystems. Exclusion analyses were completed for PKCC063, PKCC131, and PKCC168 using closely spaced STR markers. The sequences of the primer pairs used for exclusion analysis and amplification conditions are available upon request.
7
STR Analysis of Patient and hiPSC Samples
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The STR analysis was carried out by Guangzhou Cellcook Biotech Co., Ltd, China. Briefly, gDNA was extracted from PBMCs of the patient and hiPSCs with the DNeasy Blood and Tissue Kit (QIAGEN, Duesseldorf, Germany). Microsatellite DNA locus amplification was carried out by PCR using the STR Multi-Amplification Kit (PowerPlex 18D System) according to the manufacturer’s instructions. The PCR products of two multiplexes (STR loci and the sex-linked gene amelogenin) were assayed with 3100 DNA Analyzer (Applied Biosystems®). The following loci were tested: AMEL, D3S1358, D1S1656, D6S1043, D13S317, PentaE, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, and FGA. The results were analyzed by ABI Genotyper software.
8
DNA Purification from Agarose Gel
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For DNA purification from the agarose gel, the Vivantis GF-1 Nucleic Acid Extraction Kit was used and the manufacturer’s protocol was followed. DNA samples were sequenced by using Big Dye chemistry (Applied Bioscience Inc., USA). The samples were sequenced with the related forward and reverse primers using the Applied Biosystems Prism Dye Termination method according to the manufacturer’s instructions (Big Dye Deoxy Terminators; Applied Biosystems, Weiterstadt, Germany). Sequencing was performed on an automated sequencer (Applied Biosystems; 3100 DNA Analyzer). Chromatograms of sequences were obtained and submitted to GenBank. The accession numbers of the sequences are: JN315348.1, JQ394887-JQ394890, JN626460, JN626461, JN626462, JN626463, JN626464, JN626465, JN315348, JN315349, JN315350, JN315351, JN315352, JN315353, JN315354, JN315355, JN315356, and JN626466.
9
Genome-wide Linkage Mapping Using STR Markers
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Applied Biosystems MD-10 linkage mapping panels (Foster City, CA) were used to complete the genome-wide scan for family PKRP133. Multiplex PCR was completed as described previously [15 (link)]. The PCR products were mixed with a loading cocktail that contained HD-400 size standards (Applied Biosystems) and were resolved in an Applied Biosystems 3100 DNA Analyzer. Genotypes were assigned using the GeneMapper software from Applied Biosystems. Exclusion analyses were completed for PKRP140 using closely spaced short tandem repeat (STR) markers. The sequence of the primer pairs used for the exclusion analyses and amplification conditions are available upon request.
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