For confirmation of identified mutations we performed Sanger sequencing of cDNA samples. DNA-fragments were generated by PCR using the following oligonucleotides: HIST1H3B-for 5′-ATGGCTCGTACTAAACAGACAGC-3′, HIST1H3B-rev 5′-AGAGCCTTTGGGTTTTAAGACTG-3′, KDM7A-for 5′-GTAGGAATTATGTGGACAGCAG-3′, KDM7A-rev 5′-TATACACACAAACTGCTCCAGG-3′, SETD2-for 5′-CATGGACAGTGCAATCTCTGATG-3′, SETD2-rev 5′-AACTGTCCAGGAGTTTGGTGGC-3′, STAT5B-for 5′-AGGACGGAATTACACTTTCTGG-3′, STAT5B-rev 5′-ATCTGTGGCTTCACGTATCCATC-3′, TLE1-for 5′-GTGATGGTGACAAAAGCGATGAC-3′, TLE1-rev 5′-CAAAAGGAGCAGGATATGGGCC-3′. PCR products were treated using exonuclease 1 and alkaline phosphatase Illustra ExoProStar according to the recommended protocol for a 5 μl aliquot (GE Healthcare Life Sciences, Freiburg, Germany). The sequencing reactions were performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher) for 25 cycles in a Veriti Thermal Cycler (Thermo Fisher). For purification we used CleanSEQ reagent in combination with the Agencourt CleanSEQ magnetic plate (Beckman Coulter, Krefeld, Germany). The beads were eluted with 40 μl HiDi formamide and applied to the ABI 24 capillary 3500XL automated DNA sequencer (Thermo Fisher).
Illustra exoprostar
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The Illustra ExoProStar is a lab equipment product from GE Healthcare. It is designed for the isolation and purification of extracellular vesicles (EVs) from various sample types, including cell culture media and biological fluids. The product utilizes a proprietary technology to efficiently capture and purify EVs for downstream analysis and applications.
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Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (4 mentions)
Puregene dna purification kit, by Qiagen (3 mentions)
3730 dna analyzer, by Thermo Fisher Scientific (3 mentions)
Image lab software 6, by Bio-Rad (3 mentions)
Proteinase k, by Roche (3 mentions)
Sequencher 5, by Gene Codes (2 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (2 mentions)
Big dye terminator version v 1.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Sequencher, by Gene Codes (2 mentions)
Exosap it, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions)
3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Big dye terminator cycle sequencing kit 3, by Thermo Fisher Scientific (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Recombinant taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Abi 3130xl, by Thermo Fisher Scientific (1 mentions)
Qiamp dna mini kit, by Qiagen (1 mentions)
27 protocols using illustra exoprostar
1
Sanger Sequencing of Genetic Mutations
2
Genomic DNA Extraction and Amplification
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Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen; health food ingredients Nos. 3 and 4, health foods Nos. 12 and 16) or the alkaline PVPP buffer 13 (health food No. 14). The partial DNA fragments of the ITS (379 bp) and matK (143 bp) region were amplified by touchdown PCR using GoTaq Green master mix (Promega, Tokyo, Japan). The amplifying and sequencing primers for ITS were 5′-GCCACGCACTGTGTTCTCTCCT-3′ and 5′-GCAATGCTCACGGGAAGCCAACA-3′ 14 , and those for matK were 5′-CTTCGACACTGGGTGAAAGATG-3′ and 5′-AGGAACAAGAATAATCTTGG-3′ 15 (link). The PCR products were purified using Illustra ExoProSTAR (GE Healthcare, Chicago, USA) and sequenced directly with a Big Dye Terminator Cycle Sequencing Kit 3.1 on a 3130 Genetic Analyzer (Applied Biosystems, Tokyo, Japan) following the manufacturer’s instructions.
3
Multilevel S. aureus Subtyping
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All selected S. aureus isolated were subtyped using spa typing [26 (link)]. After amplification of the spa gene, PCR products were cleaned up with Illustra Exoprostar, (GE Healthcare Bio-sciences, PA, USA) then submitted to the Biomedical Genomic Center (BMGC, University of Minnesota, MN, USA) to obtain gene sequences. After aligning sequences using Sequencher 5.1 software (Gene Codes Corporation, MI, USA), each sequence was submitted to Ridom spa typing database (http://spa.ridom.de/index.shtml ).
Multi-locus sequence typing (MLST) of S. aureus was performed following the methods previously reported [27 (link)]. Briefly, seven housekeeping genes (carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glpF), guanylate kinase (gmk), phosphate acetyltransferrase (pta), triose-phosphate isomerase (tpi), and acetyl coenzyme A acetyltransferase (yqiL)) were amplified and sequenced. Specific allelic numbers of each isolate and sequence type were obtained via the MLST database of S. aureus (http://saureus.mlst.net ).
Multi-locus sequence typing (MLST) of S. aureus was performed following the methods previously reported [27 (link)]. Briefly, seven housekeeping genes (carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glpF), guanylate kinase (gmk), phosphate acetyltransferrase (pta), triose-phosphate isomerase (tpi), and acetyl coenzyme A acetyltransferase (yqiL)) were amplified and sequenced. Specific allelic numbers of each isolate and sequence type were obtained via the MLST database of S. aureus (
4
DNA extraction and gene amplification for biodiversity
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Genomic DNA was extracted from 96% ethanol-preserved tissue samples (liver, muscle tissue or scales) using a modified salt precipitation method based on the Puregene DNA purification kit (Gentra Systems). We amplified the 16S gene using the primers 16Sar-L and 16Sbr-H-R from Palumbi et al. (1991) . Additionally, the Cytb gene was obtained with the primers L14910 and H16064 developed by Burbrink et al. (2000) (link), whereas the gene coding for the subunit 4 of the NADH dehydrogenase was amplified with the primers ND4 and Leu developed by Arévalo et al. (1994) . PCR reactions contained 2 mM (Cytb and ND4) or 3 mM (16S) MgCl2, 200 µM dNTP mix, 0.2 µM (16S and Cytb) or 0.8 µM (ND4) of each primer and 1.25 U (16S and Cytb) or 0.625 U (ND4) Taq DNA Polymerase Recombinant (Thermo Fisher Scientific) in a 25 µL total volume. The nucleotide sequences of the primers and the PCR conditions applied to each primer pair are detailed in Appendix II. PCR products were cleaned with Exonuclase I and Alkaline Phosphatase (Illustra ExoProStar by GE Healthcare) before they were sent to Macrogen Inc (Korea) for sequencing. All PCR products were sequenced in both forward and reverse directions with the same primers that were used for amplification. The edited sequences were deposited in GenBank (Appendix I).
5
WFS1 and CISD2 gene exon sequencing
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The coding exons of WFS1 (NM_006005.3) and CISD2 (NM_001008388.4) were amplified with intronic primers by using standard conditions. Primers and PCR conditions are available on request. PCR products were purified with an Illustra ExoProStar enzyme (GE Healthcare, England), processed with a BidDye® Terminator Cycle Sequencing kit (Thermo Fischer, Foster City, CA, USA) and analyzed on an ABI 3130 XL automated sequencer (Applied Biosystems).
6
Genetic Analysis of Glioblastoma Specimens
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All GBM specimens were FFPE. DNA was extracted using the QIAmp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Genomic DNA amplification for TERT promoter region (260 bp) containing −124 C>T and −146 C>T mutation sites, as well as the SNP rs2853669 (−245 T>C) was carride out as previously described.30 (link) The amplified products were purified with the Illustra ExoProStar (GE Healthcare, Amersham, UK) and sequenced on a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA). All samples were analyzed in forward and reverse directions. Telomere length was determined by multiplex PCR assay.31 (link) RTL values were calculated as telomere/single-copy gene ratio, according to a previous report.32 (link) MGMT promoter methylation was assessed by pyrosequencing as described in Indraccolo et al.30 (link) IDH1/2 mutational status was assessed by immunohistochemistry or DNA sequencing.30 (link)
Genomic DNA amplification for TERT promoter region (260 bp) containing −124 C>T and −146 C>T mutation sites, as well as the SNP rs2853669 (−245 T>C) was carride out as previously described.30 (link) The amplified products were purified with the Illustra ExoProStar (GE Healthcare, Amersham, UK) and sequenced on a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA). All samples were analyzed in forward and reverse directions. Telomere length was determined by multiplex PCR assay.31 (link) RTL values were calculated as telomere/single-copy gene ratio, according to a previous report.32 (link) MGMT promoter methylation was assessed by pyrosequencing as described in Indraccolo et al.30 (link) IDH1/2 mutational status was assessed by immunohistochemistry or DNA sequencing.30 (link)
7
Sanger Sequencing for Variant Validation
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Automated Sanger sequencing was carried out to confirm variants of interest and for segregation analysis when familial samples were available. Polymerase chain reaction–amplified regions (primer sequences are available on request) were purified enzymatically with Illustra ExoProStar (GE Healthcare Life Sciences) and sequenced using a BigDye Terminator, version 3.1 kit (Thermo Fisher Scientific) on an ABI 3130x1 automated DNA sequencer (Thermo Fisher).
8
Molecular Detection and Sequencing of HfMV1
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All H. albidus and H. fraxineus isolates were screened for the presence of HfMV1 using a specific PCR assay [26 (link)]. Complementary DNA (cDNA) was produced with the Maxima First Strand cDNA Synthesis Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. For sequencing, PCR products were purified using Illustra ExoProStar (GE Healthcare Life Sciences, Pittsburgh, PA, USA) according to the instructions. Sanger sequencing of the PCR products was done on both strands using the same primers as for PCR. In addition, for 40 isolates (8 for H. albidus, 32 for H. fraxineus) the full length viral RdRP gene (2151 bp) was sequenced in both directions as described in [26 (link)]. To account for mutations at primers sites in some of the isolates, additional primers had to be designed to obtain the complete ORF (see Table S2 ).
9
Genomic DNA Extraction and PCR Amplification
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Genomic DNA was isolated by isopropanol precipitation of keratinocyte lysates (lysis buffer was Tris [pH 8] 100 mM, EDTA 5 mM, SDS 0.2%, NaCl 200 mM, and 1 mg/mL proteinase K [Roche Diagnostics, Mannheim, Germany]) and resuspended in Tris/EDTA (TE) buffer. Approximately 20–50 ng genomic DNA was used for PCR amplification. PCR fragments spanning the nuclease target sites were generated with primers F1/R (F1, 5′-gtgagtggtggctgaagcac-3′; and R, 5′-accccaccaaggaaactga-3′). PCR program TD 68-63 was as follows: 94°C for 5 min; 5 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 30 s, decreasing annealing temperature 1°C every cycle; followed by 30 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 30 s; then 72°C for 7 min. PCR products were analyzed in 1.5% agarose gel. Molecular weight marker was IX (Sigma-Aldrich). For sequencing, PCR products were treated with Illustra ExoProStar (GE Healthcare, UK), sequenced using Big Dye Terminator version (v.)1.1 Cycle Sequencing kit (Thermo Fisher Scientific, Waltham, MA) and examined on a 3730 DNA Analyzer (Life Technologies, Carlsbad, CA). Chromatograms were analyzed using Sequencher (Gene Codes, Ann Harbor, MI) and Chromas (Technelysium, Australia) software. Bio-Rad Image Lab Software 6.0 was used for PCR band densitometry.
10
S. aureus Subtyping Using spa and MLST
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All selected S. aureus isolates were subtyped using spa typing [36 (link)]. After amplification of spa, PCR products were cleaned up with Illustra Exoprostar, (GE Healthcare Bio-sciences, PA, USA) and sequenced at the University of Minnesota Genomics Center. Sequences aligned using Sequencher 5.1 software (Gene Codes Corporation, MI, USA) were submitted to the Ridom spa typing database (http://spa.ridom.de/index.shtml ).
Multi-locus sequence typing (MLST) of S. aureus was performed following methods reported previously via the MLST database of S. aureus (http://saureus.mlst.net ) [37 (link)]. MLST typing was performed purposively so that at least one isolate from each spa type detected was also evaluated by MLST.
Multi-locus sequence typing (MLST) of S. aureus was performed following methods reported previously via the MLST database of S. aureus (
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