Primer extension analysis was performed using the 5’ RACE kit (Invitrogen) according to the manufacturer’s instructions. Briefly, cDNA synthesis was performed using gene specific primers (GSP) listed in Table 1 . cDNA was purified and used in TdT reactions that added a poly-cytosine tail to the 5’ end of the cDNA. Tailed cDNA was subsequently used in PCR reactions with an internal GSP primer (ackA-GSP2 or pta-GSP2—Table 1 ) and an abridged anchor primer provided by Invitrogen. PCR products were analyzed on an agarose gel, purified on a Qiaquick PCR purification column (Qiagen) and subjected to Sanger sequencing (ACGT Inc.).
Sanger sequencing
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Sanger sequencing is a DNA sequencing method that uses chain-termination inhibitors to determine the nucleotide sequence of a DNA sample. It is a widely used laboratory technique for analyzing genetic information.
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Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (5 mentions)
Qiaquick gel extraction kit, by Qiagen (5 mentions)
Onestep rt pcr kit, by Qiagen (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (2 mentions)
Pcr buffer 1, by Roche (2 mentions)
Byqiaquick pcr purification kit, by Qiagen (2 mentions)
Pcr purification kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
5 race kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification column, by Qiagen (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Superscript 3 rt pcr, by Thermo Fisher Scientific (1 mentions)
Dnase, by Qiagen (1 mentions)
Oxoid purified agar, by Thermo Fisher Scientific (1 mentions)
Gibco opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamin 2000, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Genejet genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
Nebnext ultra dna library prep kit, by New England Biolabs (1 mentions)
22 protocols using sanger sequencing
1
Primer Extension Analysis of Gene Transcripts
2
CRISPR-Cas9 Mediated Deletion of Hsd11b1 Exon1 in Mice
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Two gRNAs flanking exon1 region of Hsd11b1 were designed using the software CRISPOR (http://crispor.tefor.net/ ). Both gRNAs were synthesized, mixed with Cas9 protein and microinjected into 30-50 mouse zygotes at the Medical Research Council transgenic facility in Imperial College London. F0 compound homozygous were crossed with wild type. F1 heterozygous mice were sequenced to determine whether exon1 is deleted. Heterozygous mice positive for exon1 deletion were then crossed to generate wild-type, heterozygous and homozygous littermates. Genotyping PCR reaction was carried out using the primer set listed in Table 1
3
Bovine CD14 Promoter Amplification
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We extracted genomic DNA from 200µL of whole blood utilizing the Isolate II Genomic DNA extraction kit (Bioline USA Inc., Swedesboro, NJ, USA), following the manufacturer’s instructions; a final volume of 100 µL eluted DNA was stored at 4 °C until use and concentration was quantified with a spectrometer (PG Instruments Ltd, England, UK). Specific primer pairs were designed with Primer Express (version 4.0) to amplify the ~1.6 kB promoter region of bovine CD14 gene (Supplementary Table S1 ). Using primer pairs and EconoTaq Plus Green 2X PCR master mix (Lucigen Corporation, Middleton, WI, USA), we amplified 1 μL of genomic DNA, optimizing reactions to a final volume of 25 μL [16 (link)]. The PCR conditions were programmed as follows: 94 °C for 2 min, and 35 cycles of 94 °C for 30 s, 59 °C for 30 s, 72 °C for 50 s, then the final extension for 5 min at 72 °C. Five microliters of amplified products were examined and band size was determined with a GeneRuler 100bp Plus DNA ladder (Supplementary Table S1 ). Amplified PCR products (n = 40) were purified with QIAquick PCR purification kit (Qiagen Inc., Valencia, CA, USA), and 2 μL of purified products were prepared for Sanger sequencing (Genewiz, South Plainfield, NJ, USA).
4
Norovirus Genotyping from UNEX Disks
5
SARS-CoV-2 S Gene Sequencing
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Reverse transcription-polymerase chain reaction (RT-PCR) was performed on virion RNA from virus stock passages using a range of primers (Table 2 ). Reactions were performed with a Superscript III First-Strand Synthesis System (Invitrogen) using a virus-specific forward primer that bound in orf1ab. The cDNAs encoding S gene sequences were amplified by PCR, either in their entirety or as four overlapping fragments, were carried out using Phusion High-Fidelity DNA Polymerase (New England Biolabs). Amplicons were agarose gel-extracted and purified using a QIAquick gel extraction kit (Qiagen) before Sanger sequencing (Genewiz, New Jersey).
6
Genetic Profiling of Meningioma
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Variants identified by WES, as well as selected regions of previously published genes implicated in meningioma (NF2, AKT1, TRAF7, SMO, and KLF4), were analyzed by PCR and Sanger sequencing. Mutant sites from NF2 (22), TRAF7 (5), AKT1 (1), SMO (2), KLF4 (1), and TERT (2) were sequenced. DNA from the four meningioma cell lines and one normal human cell line (293T) were amplified using PCR primers designed to target identified mutations (S1 Table
7
SARS-CoV-2 Genome Sequencing by RT-PCR
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Reverse transcription/polymerase chain reaction (RT-PCR) was performed on virion RNA from virus stock passages using a range of primers (Table 2 ). cDNA reactions were performed with a SuperScript III First-Strand Synthesis System (Invitrogen) using a virus-specific forward primer that bound in orf1ab. The cDNAs encoding S gene sequences were amplified by PCR, either in their entirety or as four overlapping fragments, using Phusion High-Fidelity DNA Polymerase (New England Biolabs [NEB]). Amplicons were agarose gel-extracted and purified using a QIAquick gel extraction kit (Qiagen) before Sanger sequencing (Genewiz, NJ, USA).
8
Single-Cell Polylox Sequencing Protocol
9
Single-Cell Polylox Sequencing Protocol
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For the isolation of single HSC, BM cells were stained as described
above. During FACS sorting, individual cells were deposited into 8-tube PCR
stripes containing in each well 25 μl lysis buffer (12.6 μg
proteinase K (Thermo Fisher Scientific) in PCR buffer 1 (Roche)). Lysis was done
for 1 h at 55 °C, terminated at 95 °C for 10 min, and cooled to 4
°C before adding the remaining PCR reagents to a final volume of 50
μl. The Polylox cassette was then amplified by nested
PCR. First round PCR: primer #2450 (see above) and primer #494
(5′-AGCTACAGCCTCGATTTGTGGTG-3′) for 5 min at 95 °C; (30 s
at 95 °C, 30 s at 56 °C, 5 min at 72 °C) 35 times; 10 min
at 72 °C. Second round PCR: 1-2 μl of first PCR reaction was used
as template and amplified with primers #2426
5′-CGACGACACTGCCAAAGATTTC-3′ and #2427 (see above) for 5 min at 95
°C; (30 s at 95 °C, 30 s at 62 °C, 5 min at 72 °C)
35 times; 10 min at 72 °C. The nested PCR products were purified by
QIAquick PCR Purification Kit (Qiagen), analyzed by gel electrophoresis for
product length, and analyzed by Sanger sequencing (GATC Biotech). Barcodes were
decoded for each of these sequences.
above. During FACS sorting, individual cells were deposited into 8-tube PCR
stripes containing in each well 25 μl lysis buffer (12.6 μg
proteinase K (Thermo Fisher Scientific) in PCR buffer 1 (Roche)). Lysis was done
for 1 h at 55 °C, terminated at 95 °C for 10 min, and cooled to 4
°C before adding the remaining PCR reagents to a final volume of 50
μl. The Polylox cassette was then amplified by nested
PCR. First round PCR: primer #2450 (see above) and primer #494
(5′-AGCTACAGCCTCGATTTGTGGTG-3′) for 5 min at 95 °C; (30 s
at 95 °C, 30 s at 56 °C, 5 min at 72 °C) 35 times; 10 min
at 72 °C. Second round PCR: 1-2 μl of first PCR reaction was used
as template and amplified with primers #2426
5′-CGACGACACTGCCAAAGATTTC-3′ and #2427 (see above) for 5 min at 95
°C; (30 s at 95 °C, 30 s at 62 °C, 5 min at 72 °C)
35 times; 10 min at 72 °C. The nested PCR products were purified by
QIAquick PCR Purification Kit (Qiagen), analyzed by gel electrophoresis for
product length, and analyzed by Sanger sequencing (GATC Biotech). Barcodes were
decoded for each of these sequences.
10
Genetic Analysis of ERCC6 Mutation
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WES of the proband was performed by the Beijing Genomics Institute®, and the result was interpretated by Dr. Pi-Lin Sung, a clinical geneticist at Shuang Ho hospital in Taiwan. Sequencing reads were mapped to the GRCh37/hg19 reference genome by using the Burrows–Wheeler algorithm [10 ]. The WES test report revealed the presence of a missense mutation in the ERCC6 gene due to a homozygous point mutation at the genomic level [NM_000124.2: c.1607T>G; p.Leu536Trp].
Sanger sequencing of the proband and his parents was performed to determine if this mutation was inherited from the parents or were caused by a sporadic event. The DNA encoding portion of the ERCC6 gene was PCR amplified from the genomic DNA using the following primer pairs: ERCC6 (c.1607T>G, p.Leu536Trp) F: CTGCCCTACAGCTCCATT and R: TCCACCATTTGCCATTTT (PCR product: 386 bp). The resulting PCR products were purified using QIAquick PCR Purification Kit (Qiagen®) and then subjected to Sanger sequencing (Genomics®, Taipei, Taiwan).
Several bioinformatics databases were used to interpret the pathogenicity of this mutation in the ERCC6 gene, including Varsome, Uniport, ClinVar, ClinVar Miner, and dbSNP. Furthermore, the allele frequency of the variant was investigated in the Taiwan BioBank as a local database and in the Genome Aggregation Database (gnomAD) as a global database.
Sanger sequencing of the proband and his parents was performed to determine if this mutation was inherited from the parents or were caused by a sporadic event. The DNA encoding portion of the ERCC6 gene was PCR amplified from the genomic DNA using the following primer pairs: ERCC6 (c.1607T>G, p.Leu536Trp) F: CTGCCCTACAGCTCCATT and R: TCCACCATTTGCCATTTT (PCR product: 386 bp). The resulting PCR products were purified using QIAquick PCR Purification Kit (Qiagen®) and then subjected to Sanger sequencing (Genomics®, Taipei, Taiwan).
Several bioinformatics databases were used to interpret the pathogenicity of this mutation in the ERCC6 gene, including Varsome, Uniport, ClinVar, ClinVar Miner, and dbSNP. Furthermore, the allele frequency of the variant was investigated in the Taiwan BioBank as a local database and in the Genome Aggregation Database (gnomAD) as a global database.
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