We analyzed 2 single-nucleotide polymorphisms (SNPs) with the Multiplex SNaPshot kit for rs5498 T>C in exon 6 of the ICAM1 gene and rs1041163 T>C in the VCAM1 gene. The primers for ICAM-1 rs5498 were: Forward: 5′-GTAAAGGACCTCTGGGTTACT-3′ Reverse: 5′-AGGCTTCTGGATCTCCTCTT-3′. The primers for VCAM-1 rs1041163 were: Forward: 5′-ACTCACAGAGCACATTCACG-3′ Reverse: 5′-AGATCTTGAGGGCACCTAC-3′. The PCR protocol was: initial denaturation at 94°C for 5 min, 33 cycles of amplification at 94°C for 30 s, annealing at 55°C for 30 s, and extension for 30 s at 72°C, followed by a final extension for 5 min at 72°C. PCR products were purified with Shrimp Alkaline Phosphatase (SAP) and preserved at 4°C. The primers and sequences of the SNaPshot extension reaction were: rs5498-R-R-60: 5′-tttttttttttttttttttttttttttttttttttttttttAGCACATTCACGGTCACCT-3′. rs1041163-Y-F-34: 5′-tttttttttttGCTAGTATTTCCTGAATCAATTT-3′. The reaction protocol was: 25 cycles of extension at 96°C for 10 s, at 51°C for 5 s, and at 60°C for 30s, followed by 1 cycle at 4°C. The products were purified with 0.3 μl SAP and 1.7 μl ddH2O and stored at 4°C. Analysis using ABI 3730XL sequencer was: 1 μl product, 0.2 μl Genescan™-120LIZ Size Standard and 8.8 μl deionized formamide were mixed in a 96-well-plate, denatured at 95°C for 5 min, and directly analyzed by use of an ABI 3730XL sequencer.
3730xl sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, France
The 3730XL sequencer is a high-throughput genetic analysis instrument designed for DNA sequencing. It utilizes capillary electrophoresis technology to analyze DNA samples and generate sequencing data. The 3730XL is capable of processing multiple samples simultaneously, providing efficient and reliable genetic information for research and diagnostic applications.
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Lab products found in correlation
Plant genomic dna kit, by Tiangen Biotech (6 mentions)
High pure pcr product purification kit, by Roche (5 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (3 mentions)
Qiaquick gel extraction kit, by Qiagen (3 mentions)
2720 thermal cycler, by Thermo Fisher Scientific (2 mentions)
Big dye terminator kit v3, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Primer premier 5, by Premier Biosoft (2 mentions)
Pcr master mix, by Aidlab (2 mentions)
Abi prism bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Qiaamp blood kit, by Qiagen (2 mentions)
Accuprime pfx dna polymerase, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Genemapper 4, by Thermo Fisher Scientific (1 mentions)
Snpscan genotyping assay, by Genesky Biotechnologies (1 mentions)
Dna purification kit, by Promega (1 mentions)
Phusion high fidelity buffer, by New England Biolabs (1 mentions)
Phusion taq, by New England Biolabs (1 mentions)
Cobas amplicor monitor, by Roche (1 mentions)
138 protocols using 3730xl sequencer
1
Multiplex SNaPshot Genotyping of ICAM1 and VCAM1 SNPs
2
Purification and Sequencing of PCR Products
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The second round of PCR was performed with inner primers and PCR products were purified using the High Pure PCR Product Purification Kit (Roche Diagnostic, Mannheim, Germany) according to the manufacturer’s instructions and were used for direct sequencing using the dye termination method and an ABI 3730xl sequencer (19 (link)).
3
Molecular Identification of Alternaria Strain
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Strain SPS-2 was inoculated into the PDA medium for a few days to culture and become cultivated, followed by morphological observations and the nuclear ribosomal internal transcribed spacer (ITS) gene amplicon sequencing. First, the sequence of ITS was amplified via polymerase chain reaction (PCR) with ITS1-ITS4 primer pairs. Additionally, 3 µL of the PCR product was taken for 1% agarose gel electrophoresis detection to confirm the PCR amplification fragment. Then, the products were recovered using an AxyPrep DNA Recovery Kit. Finally, sequencing was performed with an ABI3730-XL sequencer. Meanwhile, the ITS sequence with 1199 bp was submitted to GenBank in the NCBI database, and an accession number was acquired. A phylogenetic tree was constructed using MEGA (version 7.0, https://www.megasoftware.net/ , accessed on 25 June 2022) by comparison with other Alternaria strains with high similarities in the NCBI database.
4
Genotyping of miRNA-146a rs2910164
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Genomic DNA was extracted from the peripheral blood samples collected in EDTA test tubes using a DNA Purification Kit (Promega, Madison, WI, USA). SNPscan™ genotyping assay (Genesky Biotechnologies Inc., Shanghai, China) was used to analyze the genotyping of miRNA-146a rs2910164 C>G polymorphism. In brief, a 150 ng DNA sample was heated to 98°C and held for 5 minutes. The ligation reaction was carried out in an ABI 2720 thermal cycler. Then, a 48-plex fluorescence polymerase chain reaction (PCR) was conducted. In an ABI 3730XL sequencer, capillary electrophoresis was harnessed to analyze the PCR products. GeneMapper 4.1 software (Applied Biosystems, Foster City, CA, USA) was used to read the information of the genotype. For quality control, different technicians genotyped 4% of the genomic DNA samples that were randomly selected. And, the results were in full accord with the findings of the first assays.
5
SNP Identification from Blood Samples
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DNA was extracted from the blood samples using a DNA extraction kit, and forward and reverse primers were designed for SNP determination and then amplified by polymerase chain reaction (PCR) (9700 PCR Amplifier, ABI Corporation, CA, USA). The PCR products were verified by 1% agarose electrophoresis (150 V, 100 mA for 20 minutes).
Gene sequencing was performed using Sanger sequencing. After the PCR products were purified, the samples were run on a 3730XL sequencer (ABI), and Run 3730 data collection V3.0 software was run at the same time. The final sequencing results were assessed using Chromas or SeqMan software, and sequence alignment was completed using SeqMan software.
Gene sequencing was performed using Sanger sequencing. After the PCR products were purified, the samples were run on a 3730XL sequencer (ABI), and Run 3730 data collection V3.0 software was run at the same time. The final sequencing results were assessed using Chromas or SeqMan software, and sequence alignment was completed using SeqMan software.
6
Validating Structural Variants in Arabidopsis
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Structural variants in Ws-2 relative to the Col-0 reference were predicted using Pindel (Ye et al. 2009 (link)). For validation, we PCR amplified 1−2 kb of sequence spanning the predicted CO breakpoints. For the Ws-2 chromosome 4 insertion, we amplified ~500 bp to 1 kb around the predicted breakpoints. Genomic DNA was PCR amplified using reactions containing 0.4 µM forward and reverse primers, 0.2 µM dNTPs, 1x Phusion High Fidelity Buffer, and 0.2 units Phusion Taq (New England Biolabs) along with template DNA and incubated in an MJ Research PTC-200 or PTC-225 thermal cycler (Hercules, CA) at 94° for 2 min, followed by 37 cycles of 20 sec at 94°, 20 sec at 50−60°, and 30 sec at 72° with a final extension step at 72° for 5 min. Primer sequences are provided in Table S2 . We sequenced the amplified regions in the Col-0 and Ws-2 parents and the F2 individuals with the predicted CO in sequencing by assembling reactions containing 1 µM primer, 0.5 µL of BigDye Terminator ready reaction mix, and 1X sequencing buffer and incubating for 20s at 96°, followed by 29 cycles of 10 sec at 50° and 4 min at 60° before analyzing on an ABI 3730xL sequencer.
7
SSR Molecular Marker Screening Protocol
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The SSR molecular marker test includes DNA extraction, primer screening, fluorescence quantitative PCR, and electrophoresis tube detection. DNA was extracted using a genomic kit from Beijing Botanical Biofet Plant. This DNA extraction kit was used to select primers as the 36 main reference pairs [21 ,22 ,23 (link)], and 12 pairs of primers with high and stable polymorphism were screened (Table 2 ) for fluorescence quantitative PCR and electrophoresis tube detection.
The fluorescence quantitative PCR consists of a 20 µL system, including 17 µL of gold mix (green), 1 µL of front primers, 1 µL of back primers, and 1 µL of DNA template. The amplification procedures are listed as follows. First, pre-denaturation was performed at 98 °C for 2 min. The second stage was the cycle stage. Samples were denatured at 98 °C for 10 s, annealing at 55–62 °C for 10 s, extended at 72 °C for 10 s, and cycled 35 times. The third stage was the extension stage. Samples were extended at 72 °C for 5 min. For the capillary test, the mixing plate was heated at 95 °C for 5 min with a metal bath heater and immediately put into an ice box at −20 °C. The mixing plate was removed after cooling, centrifuged at 4000 rpm, thawed, and mixed well. Then, it was placed in an ABI 3730xl sequencer for capillary electrophoresis.
The fluorescence quantitative PCR consists of a 20 µL system, including 17 µL of gold mix (green), 1 µL of front primers, 1 µL of back primers, and 1 µL of DNA template. The amplification procedures are listed as follows. First, pre-denaturation was performed at 98 °C for 2 min. The second stage was the cycle stage. Samples were denatured at 98 °C for 10 s, annealing at 55–62 °C for 10 s, extended at 72 °C for 10 s, and cycled 35 times. The third stage was the extension stage. Samples were extended at 72 °C for 5 min. For the capillary test, the mixing plate was heated at 95 °C for 5 min with a metal bath heater and immediately put into an ice box at −20 °C. The mixing plate was removed after cooling, centrifuged at 4000 rpm, thawed, and mixed well. Then, it was placed in an ABI 3730xl sequencer for capillary electrophoresis.
8
Germline Mutation Analysis of MSH6 Gene
9
Blinded HIV Viral Load and Genotyping
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VLs were assayed blind to randomised group using Abbott m2000sp/rt or Roche COBAS Amplicor Monitor ultrasensitive tests, v1.5, with lower limit of detection of 80 copies/ml because many samples were diluted 1:2 due to low volumes. Genotyping for reverse transcriptase was done blind to randomised group using an in-house assay at a WHO-designated laboratory (Joint Clinical Research Centre, Kampala, Uganda) using the ABI 3730xl sequencer. Major NRTI and NNRTI mutations were defined using IAS-USA 2013 [11 (link)], and drug susceptibility was predicted using the Stanford algorithm version 7 [12 (link)].
10
Sanger Sequencing for Variant Validation
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Candidate gene mutations were validated using conventional Sanger sequencing methods. The primers were designed using the online Primer3 software. The PCR products were purified by shrimp alkaline enzyme (SAP) (from Promega) and exogenous enzyme I (EXO I) (from Epicentre) and sequenced using a BigDye® Terminator Cycle Sequencing Ready Reaction Kit, version 3.1 (Foster, CA, USA). An ABI 3730xl sequencer (Carlsbad, CA, USA) was used to analyze the sequencing products. The sequencing documents were analyzed by PolyPhred software.
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