Virus RNA from six qRT-PCR positive samples selected according to lowest cycle threshold (Ct) values, was reverse-transcribed using the TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific Life Technologies GmbH, Darmstadt, Germany) with a mixture of random hexamers and cDNA were amplified following the manufacturer’s instructions. Previously published primers31 (link) were synthesised by GenExpress and used for the generation of the near full-length sequence. Amplification was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, USA) following the manufacturer’s instructions. Amplified libraries were sequenced directly using the BigDye version 3.1 cycle sequencing kit (Thermo Fisher Scientific Life Technologies GmbH), and data were subjected to an automated sequence analysis using a Genetic Analyser 3500 xl Dx (Thermo Fisher Scientific Inc., USA). Sequence and phylogenetic analyses were conducted using the MEGA software version 6.0636 (link). Maximum likelihood trees were inferred for 1000 replications using the General Time Reversible model with 4 Gamma categories. Nodes were supported by the bootstrap values and values below 70% are hidden for more clarity.
Big dye version 3.1 cycle sequencing kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
The Big Dye Version 3.1 Cycle Sequencing Kit is a laboratory reagent used for DNA sequencing. It contains the necessary components, including fluorescently labeled dideoxynucleotides, to perform cycle sequencing reactions. The kit enables the determination of the nucleotide sequence of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 310 automated sequencer, by Thermo Fisher Scientific (3 mentions)
Lightcycler multiplex rna virus master, by Roche (2 mentions)
Seqstudio genetic analyser, by Thermo Fisher Scientific (2 mentions)
Nucleospin gel and pcr clean up, by Macherey-Nagel (2 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Bigdye xterminator purification kit, by Thermo Fisher Scientific (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Transit x2, by Mirus Bio (1 mentions)
Silencer select, by Thermo Fisher Scientific (1 mentions)
Kod one, by Toyobo (1 mentions)
Kod sybr, by Toyobo (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Protoscript 2 reverse transcriptase, by New England Biolabs (1 mentions)
Applied biosystems 3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Kod fx neo, by Toyobo (1 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Smarter race cdna amplification kit, by Takara Bio (1 mentions)
Genotyping console software, by Thermo Fisher Scientific (1 mentions)
16 protocols using big dye version 3.1 cycle sequencing kit
1
Near Full-Length SARS-CoV-2 Genome Sequencing
2
SARS-CoV-2 Spike Protein Variant Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The spike protein variants H69V70del, Y144del, T478K, K417N/T, P681R/H, E484K, and N501Y were identified using a RT-PCR melting curve analysis targeting amino acid mutations. The VirSNiP SARS-CoV-2 typing assays (TibMolBiol, Cat.-No. 53-0781-96, 53-0799-96, 53-0807-96, 53-0811-96, 53-0813-96) and LightCycler® Multiplex RNA Virus Master (Roche Cat.-No. 06 754 155 001) were performed following the manufacturer’s instructions. The six pairs of forward (F) and reverse (R) sequencing primers were designed according to the SARS-CoV-2 reference strain NC_045512, resulting in overlapping fragments covering 2.1 kb of the spike-gene (Supplementary Table S1 ). The amplification conditions were as follows: RT at 55 °C for 5 min, activation for 2 min at 95 °C, 45 cycles with 10 s at 95 °C, 20 s at 58 °C, and 30 s at 72 °C for product amplification. The amplified products with 411–501 bp were purified with NucleoSpin Gel and PCR clean-up (Macherey-Nagel, Düren, Germany), sequenced using the BigDye version 3.1 cycle sequencing kit (Thermo Fisher Scientific Life Technologies GmbH, Waltham, MA, USA), and subjected to an automated sequence analysis using the SeqStudio Genetic Analyser (Thermo Fisher Scientific Inc., Waltham, MA, USA).
3
SARS-CoV-2 Spike Protein RBD Mutation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The amino acid mutations in the spike protein RBD region 319 to 541 were identified by post-RT-PCR melting curve analysis targeting amino acid mutations R346T, S371 F/L, S373P, K444T, V445P, N460K, E484A, F486S. The VirSNiP SARS CoV-2 typing assays (TibMolBiol, Cat. No. 53-0844-96, 53-0831-96, 53-0846-96, 53-0845-96, 53-0847-96, 53-0848-96) and LightCycler® Multiplex RNA Virus Master (Roche Cat. No. 06,754,155 001) were performed following the manufacturer's instructions. The pairs of forward (F) and reverse (R) sequencing primers were designed according to the SARS-CoV-2 reference strain NC_045512, resulting in overlapping fragments covering 2.1 kb of the spike-gene [17 ]. The amplification conditions were as follows: RT at 55 °C for 5 min, activation for 2 min at 95 °C, 45 cycles with 10 s at 95 °C, 20 s at 58 °C, and 30 s at 72 °C for product amplification. The amplified products with 411–501 bp were purified with NucleoSpin Gel and PCR clean-up (Macherey-Nagel, Düren, Germany), sequenced using the BigDye version 3.1 cycle sequencing kit (Thermo Fisher Scientific Life Technologies GmbH, Waltham, MA, USA), and subjected to an automated sequence analysis using the Sanger method on SeqStudio Genetic Analyser (Thermo Fisher Scientific Inc., Waltham, MA, USA). Sequence alignments and analysis were made using the molecular evolutionary genetic analysis (MEGA) software.
4
Efficient Genomic DNA Isolation and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from hESCs or HCT116 cells using the DNeasy Blood and Tissue Kit (QIAGEN), and endpoint PCRs were performed using KOD One (TOYOBO). Sanger sequencing was performed with a cycle sequencing reaction using the BigDye, version 3.1, Cycle Sequencing Kit (Thermo Fisher Scientific) and analyzed with an ABI 3730XL DNA Analyzer (Thermo Fisher Scientific).
For knockdown experiments, siRNAs targeting OCT4 (Thermo Fisher Scientific, Silencer Select, s10872) and SOX2 (Thermo Fisher Scientific, Silencer Select, s13294) were used. Transfection of siRNAs was performed using TransIT X2 (Mirus Bio) following the manufacturer’s protocol. Seventy-two hours after transfection, cells were collected for RNA preparation.
RNA was extracted from hESCs or HCT116 cells using the RNeasy Kit (QIAGEN) and reverse transcribed using ProtoScript II Reverse Transcriptase (New England BioLabs). qRT-PCR was performed using KOD SYBR (TOYOBO) in a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific). Relative expression levels were calculated by the ΔΔCt method using ACTB as an internal control gene. Primer sequences are listed inSupplemental Table 3 .
For knockdown experiments, siRNAs targeting OCT4 (Thermo Fisher Scientific, Silencer Select, s10872) and SOX2 (Thermo Fisher Scientific, Silencer Select, s13294) were used. Transfection of siRNAs was performed using TransIT X2 (Mirus Bio) following the manufacturer’s protocol. Seventy-two hours after transfection, cells were collected for RNA preparation.
RNA was extracted from hESCs or HCT116 cells using the RNeasy Kit (QIAGEN) and reverse transcribed using ProtoScript II Reverse Transcriptase (New England BioLabs). qRT-PCR was performed using KOD SYBR (TOYOBO) in a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific). Relative expression levels were calculated by the ΔΔCt method using ACTB as an internal control gene. Primer sequences are listed in
5
Amhr2 Gene Sequence Amplification and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DNA containing exon 9 of the Amhr2 gene and the flanking region was amplified as reported previously [26 (link)] with the exception that the primers, SD3exon8-2F (5’- TGGCTCCCAGCTCAGATTC-3 ’) and SD3exon10-2R (5’-TGCGTCCTGTGCGATTT-3’ ) were used. The DNA sequence of the PCR product was directly determined by Sanger’s method from both strands using Applied Biosystems 3130 genetic analyzer (Thermo Fisher) and BigDye version 3.1 Cycle Sequencing Kit (Thermo Fisher). The DNA containing the full-length coding region of T. niphobles Amhr2 gene was amplified by PCR using KOD FX Neo (Toyobo) reagents suitable for amplifying large genomic fragments. Approximately 13 ng of genomic DNA was used as a template in 10 μl PCR reaction with the primer pair 33-1464k340F (5’-CTTCTCCAGTCTTTACCAGGAGTTTTACTT-3’ ) and 33-1464k13469R (5’-GCTCACAGAACCCTTCTCCTTTGTCTT-3’ ) (500 nM each). The cycling conditions were as follows: 94°C for 5 minutes, followed by 36 cycles of 94°C for 10 sec and 70°C for 12 min. The DNA sequence of the PCR product was directly determined using primers listed in S1 Table .
6
Full-Length cDNA Cloning from Diverse Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After extracting coding sequence information from the GenBank and in-house cDNA databases, 5′- or 3′-RACE was performed using the SMARTer RACE cDNA Amplification Kit (Clontech) to obtain full-length cDNA clones of the coding sequences. Total RNA was extracted from dormant axillary buds, shoot apices, flower buds, or roots (Magtration®; Precision System Science Co., Ltd.). Amplified fragments were sequenced using the BigDye® (version 3.1) Cycle Sequencing Kit (Thermo Fisher Scientific Inc.) and the 3730xl DNA Analyzer (Thermo Fisher Scientific Inc.). Standard procedures were used for DNA manipulations [31 ]. Information regarding the obtained sequences is provided in Supplemental Table S4 .
7
Genetic Screening for Autosomal Recessive Disorders
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gene mutations were screened by CHIPS (CEL nuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining) technology [Niida et al., 2012a [Niida et al., , 2015] ] Uniparental Isodisomy Patterns that Cause Autosomal Recessive Disorders Cytogenet Genome Res 2018;154:137-146 DOI: 10.1159/000488572 139 and confirmed by direct sequencing with an ABI 3130xl Genetic Analyser and BigDye version 3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). For patient 3, the SNRPN methylation test was performed according to an established protocol [Kosaki et al., 1997] with some modifications. Chromosomal analysis was conducted on trypsin and G-banded metaphases according to established guidelines. For patient 1, DNA microarray analysis was performed using a CytoScan 750K Array (Affymetrix) and analyzed using Chromosome Analysis Suite (ChAS) 2.1 Software (Affymetrix). Patients 2 and 3 were analyzed using the Genome-Wide Human Single Nucleotide Polymorphism (SNP) 6.0 Array (Affymetrix) with Genotyping Console Software (Affymetrix). In all cases, segments which included at least 5 consecutive probes and which were at least 50 kb in size were selected.
8
Variant Confirmation by Sanger Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All variants identified by whole exome sequencing were confirmed by Sanger sequencing of the genomic DNA extracted from the blood of patients. Primer pairs encompassing the individual sequence variants were designed for Sanger sequencing. PCR reactions were carried out in a total volume of 50 μl using GoTag® Green Mastermix (Promega). The conditions for PCR included an initial denaturation at 95 °C for 4 min, 40 cycles consisting of a denaturation at 95 °C for 30 s, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min followed by a final extension for 5 min at 95 °C. Approximately 5 μl of the reaction product was run on a 1% agarose gel containing ethidium bromide (100 mg/ml). The PCR products were purified by a standard protocol and sequenced using the Big Dye® Version 3.1 Cycle Sequencing kit (Applied Biosystems) after confirming the sizes of the amplicons.
9
Cycle Sequencing and Capillary Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following manufacturer’s instruction, the cycle sequencing of the column-purified PCR product was performed using the Big Dye Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, UK). Thereafter, the cycle sequencing product was purified using the BigDye® XTerminator™ purification kit (Applied Biosystems) and the manufacturer’s protocol was followed for the purification steps. Finally, the purified cycle sequencing PCR product was subjected to capillary sequencing in an ABI PRISM 310 Automated Sequencer (Applied Biosystems).
10
Sanger Sequencing for Variant Confirmation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For confirmation of the HRM melt curve results, Sanger sequencing was performed for all samples as it has been reported that some G6PD variants might generate melting patterns that are similar to those generated by the wild type allele [44 (link), 45 (link)]. Sequencing of G6PD gene was done using our previously published primers [26 (link)]. For sequencing, the extracted DNA samples were subjected to polymerase chain reaction (PCR) following aforementioned study protocol. Following completion of reaction cycles, PCR product purification was performed using the MinElute® PCR purification kit (Qiagen) according to manufacturer’s instruction.
The purified PCR product was subjected to chain termination reaction for Sanger sequencing using the Big Dye Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) following manufacturer’s instruction. Thereafter, the chain termination product was purified using the BigDye® XTerminator™ purification kit (Applied Biosystems). Finally, the purified chain termination PCR product was subjected to capillary sequencing in an ABI PRISM 310 Automated Sequencer (Applied Biosystems).
The purified PCR product was subjected to chain termination reaction for Sanger sequencing using the Big Dye Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) following manufacturer’s instruction. Thereafter, the chain termination product was purified using the BigDye® XTerminator™ purification kit (Applied Biosystems). Finally, the purified chain termination PCR product was subjected to capillary sequencing in an ABI PRISM 310 Automated Sequencer (Applied Biosystems).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!