To further confirm the presence of the alternative alleles in pSNMs by another independent validation platform, all the pyrosequencing-validated sites were Sanger sequenced in individual clones selected from TA-cloned PCR amplicons. The genomic DNA was amplified using primers flanking these sites (Supplementary information, Table S7 ) and the PCR products were purified. The amplicons were cloned into the Trans1-T1 phage resistant chemically competent cells using the pEASY-T1 Simple Cloning Kit (Transgen Biotech, Beijing, China). The DNA from the positive colonies was PCR amplified using the M13 universal primers, and then the purified products were sequenced using the Applied Biosystems 3730 DNA Analyzer (Life Technologies, Carlsbad, CA, USA). All the pSNMs were confirmed by the independent validation of at least two reference and mutation calls each by Sanger sequencing. We also sequenced the original PCR amplicons in both directions in the Applied Biosystems 3730 DNA Analyzer (Life Technologies).
Applied biosystems 3730 dna analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems 3730 DNA Analyzer is a fully automated capillary electrophoresis instrument designed for high-throughput DNA sequencing. It features 48 or 96 capillaries and can generate up to 384 sequencing reads per run.
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Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (7 mentions)
Genescan 500 liz size standard, by Thermo Fisher Scientific (4 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (3 mentions)
Dna purification kit, by Zymo Research (2 mentions)
Genemapper v4, by Thermo Fisher Scientific (2 mentions)
Mastercycle epgradient, by Eppendorf (2 mentions)
Biophotometer, by Eppendorf (2 mentions)
Red taq, by Thermo Fisher Scientific (2 mentions)
Peasy t1 simple cloning kit, by Transgene (1 mentions)
Takara ex taq dna polymerase, by Takara Bio (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Abi prism bigdye terminator v 3, by Thermo Fisher Scientific (1 mentions)
Platinum pcr supermix high fidelity, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Genomic dna purification kit, by Promega (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Superscript iitm reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
Minelute gel extraction kit, by Qiagen (1 mentions)
47 protocols using applied biosystems 3730 dna analyzer
1
Validating Allelic Variants through Sanger Sequencing
2
Amplification and Sequencing of rplF Gene
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The rplF fragment was amplified using the PCR primers rplF-F (5′-CAGTGACTGTTCCCGCTGGTGT-3′) and rplF-R (5′-AGGYTCAGGAGKWCGGAAHG-3′), which were designed using the primer-BLAST tool (15 (link)) available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ ) and MEGA5 (11 (link)). For PCR amplification of the rplF gene fragment, reaction mixes were incubated for 35 cycles; each cycle consisted of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. PCR products were purified using a precipitation method (16 (link)) and the nucleotide sequences of the purified PCR products were determined on each DNA strand using the primers described above by cycle sequencing with Applied Biosystems BigDye ready reaction mix (Life Technologies), used in accordance with the manufacturer's instructions. Sequence termination reaction products were separated and the sequence data collected using an Applied Biosystems 3730 DNA analyzer (Life Technologies). Nucleotide sequence data from forward- and reverse-strand electropherograms were assembled into single contiguous sequences using SeqSphere (http://www.ridom.de/seqsphere/ ) and checked using the Staden suite of programs (17 (link)).
3
MLPA Analysis for Copy Number Validation
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To rule out potential copy number abnormalities at the candidate pSNM sites, MLPA was performed on the case sample and a reference control sample obtained from an unrelated individual in whom no mosaicism was observed at the corresponding site. A pair of custom synthetic probes was designed for each validated pSNM to target its flanking regions; the distance to the pSNMs varied from 5 to 1 267 bp (Supplementary information, Table S8 ). The steps of probe preparation, ligation, and PCR amplification were performed using the EK1-FAM Probe Kit and the P300-100R Reference Probemix (MRC-Holland, Amsterdam, the Netherlands), following the manufacturer's protocol. The PCR products were analyzed on the Applied Biosystems 3730 DNA Analyzer (Life Technologies), and the signal processing, normalization and comparison were performed using Coffalyser.NET software (MRC-Holland). Each MLPA experiment on the reference sample was repeated three times. The genomic copy number analysis was reported as normal when the ratio of the normalized peak areas between the case and reference samples was 0.7-1.3, which were the default parameters on Coffalyser.NET.
4
Sanger Sequencing of Cancer Genes
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Sanger sequencing was used as a routine reference method for detecting mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. For the identification of mutations in samples with low percentages of mutant DNA, we used LNA PCR clamp amplification followed by direct Sanger sequencing. The primers and LNA-oligomers for the sequencing analysis are shown in Supplementary Tables 4 and 3 , respectively. For the amplification of most loci, we used the same primers that were used in the first-round of PCR for the biochip analysis.
PCR products were purified by ethanol precipitation of DNA with ammonium acetate and sequenced with the BigDyeTM Terminator 3.1 Cycle Sequencing Kit (Life Technologies Corporation, Carlsbad, USA) on an Applied Biosystems 3730 DNA Analyzer (Life Technologies Corporation, Carlsbad, USA) according to the manufacturer's instructions. The sequencing results were interpreted using Chromas Lite software V.2.1 (Technelysium Pty, Helensvale, Australia).
PCR products were purified by ethanol precipitation of DNA with ammonium acetate and sequenced with the BigDyeTM Terminator 3.1 Cycle Sequencing Kit (Life Technologies Corporation, Carlsbad, USA) on an Applied Biosystems 3730 DNA Analyzer (Life Technologies Corporation, Carlsbad, USA) according to the manufacturer's instructions. The sequencing results were interpreted using Chromas Lite software V.2.1 (Technelysium Pty, Helensvale, Australia).
5
Validating HBV Integration Breakpoints
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PCR and Sanger sequencing were used to verify the selected HBV integration breakpoints from HIVID. All the samples used for SANGER validation are left-over samples from previous rounds of the capture approach. PCR primers were designed based on the paired-end assembled fragment, in which one primer located in human genome and the other in HBV genome. PCR were performed by GeneAmp PCR System 9700 thermal cycler and then preceded to Sanger sequencing on Applied Biosystems 3730 × DNA analyzer (Life Technologies, Inc.).
6
Validation of Fusion Breakpoints by PCR and Sanger Sequencing
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The selected fusion breakpoints (human-human and HPV-human) were validated by PCR in bulk HeLa S3 cDNA, DNA and single-cell cDNA and DNA. We designed the PCR primers on the basis of the paired-end assembled fragments, in which one primer was located in the left gene of fusion and the other in the right gene of the fusion. Sanger sequencing was then used for the PCR validated products on an Applied Biosystems 3730 DNA analyzer (Life Technologies, Inc.). For fusion transcripts frequencies validation (RPS6KB1-VMP1 and CEP89-PEPD), 8 single cell cDNA were used for PCR validation. For HPV-host fusions, qPCR was used in additional 64 single-cell products to further validate the breakpoints frequency (Additional file 1 : Figure S1).
7
Genome Sequencing of Oshima 5-10 Strain
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Viral RNA of each Oshima 5-10 clone was extracted from the cell culture fluids of each stock virus using QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer’s protocol. Reverse transcription was performed using Superscript III reverse transcriptase (Invitrogen) and random hexamers. PCR was performed to cover the whole genome sequence using TAKARA Ex Taq DNA polymerase (TAKARA BIO Inc.). The cycle sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies), and the DNA sequence was determined in the Applied Biosystems 3730 DNA Analyzer (Life Technologies).
Primers were designed based on the genome of Oshima 5-10 strain (GenBank Acc. No. AB062063.2). Sequence was performed in both directions with specific primer corresponding to each fragment. Sequence results were analyzed with MEGA5 [29 (link)] and Unipro Ugene [30 (link)]. The nucleotides sequences were aligned by ClustalX 2.1 [31 (link)] for full genome analyses.
Primers were designed based on the genome of Oshima 5-10 strain (GenBank Acc. No. AB062063.2). Sequence was performed in both directions with specific primer corresponding to each fragment. Sequence results were analyzed with MEGA5 [29 (link)] and Unipro Ugene [30 (link)]. The nucleotides sequences were aligned by ClustalX 2.1 [31 (link)] for full genome analyses.
8
Validating CO Event Identification
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To test the correctness of our approach of CO event identification we randomly selected 24 CO regions in 15 F1 and sequenced them using Sanger technology. Based on alignment of the F1 genome and parental genomes, we designed primers that amplify short (300–600 bp) regions containing putative COs. Primer sequences are listed in the supplementary table S4 , Supplementary Material online. Polymerase chain reaction (PCR) was run on a MJ Mini thermal cycler (MJ Research, USA) using the following program: Initial denaturation –95 °C for 3 min, then 95 °C for 15 s, 61C for 30 s, and 72 °C for 30 s, 35 cycles in total. The PCR products were purified using DNA Cleanup Standard kit (Evrogen, Russia) and sequenced using ABI PRISM BigDye Terminator v. 3.1 on an Applied Biosystems 3730 DNA Analyzer (Life Technologies, USA). The resulting sequences completely matched with the assembled genomes, confirming all 24 COs.
9
Expressing MeCP2 Variants in E. coli
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MeCP2 variants from isoform were expressed in E. coli using a pET30b plasmid. The different protein variants were obtained by inserting appropriate substitutions: MBD, MBD R106W, MBD R133C, NTD-MBD-ID, NTD-MBD-ID R106W, and NTD-MBD-ID R133C (Supplemental Figure S1 ). An N-terminal polyhistidine-tag was inserted for quick purification, and it was removed through an inserted PreScission Protease cleavage site. Appropriate expression was assessed by sequencing analysis: Sanger sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA) in an Applied Biosystems 3730/DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).
10
Detecting B-Raf Mutations in Liver Tumors
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To detect B-Raf mutations in liver tumors, we performed direct sequencing as previously published. In brief, DNA was extracted from frozen materials with the DNeasy Blood and Tissue Kit (Qiagen). The region that included the B-Raf codon 637 was amplified by PCR using appropriate primers (forward: 5′-gacctcacggtaaaaataggtgac-3′; reverse: 5′-gcaattatgcctggcttacaa-3′) and Platinum PCR SuperMix High Fidelity (Invitrogen). The PCR products were purified with the Genomic DNA Purification Kit (Promega) and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) on an Applied Biosystems 3730 DNA Analyzer (Thermo Fisher Scientific).
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