Genomic DNA was isolated from blood samples using a QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Genotyping for TLR9 (−1237T/C, rs5743836; −1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) SNPs were performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as we described elsewhere [10 (link)]. The reaction was performed in a Veriti 96 Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The concentration and purity of DNA were determined using a NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The digested fragments were analyzed using a QIAxcel system (Qiagen). The selected samples of each TLR9 SNP were sequenced using the 96-capillary 3730xl DNA Analyzer (Applied Biosystems) to confirm the detected genotypes.
96 capillary 3730xl dna analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 96-capillary 3730xl DNA Analyzer is a laboratory instrument designed for high-throughput DNA sequencing. The core function of this product is to perform automated DNA fragment analysis using capillary electrophoresis technology.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna blood mini kit, by Qiagen (2 mentions)
Nanodrop 2000c uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Qiaxcel system, by Qiagen (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Instaclone pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Exicycler 96, by Bioneer (1 mentions)
Bigdye terminator kit, by Thermo Fisher Scientific (1 mentions)
Ultraclean pcr clean uptm kit, by Qiagen (1 mentions)
Abi bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
9 protocols using 96 capillary 3730xl dna analyzer
1
TLR9 SNP Genotyping from Blood
2
Genomic DNA Extraction and Sequencing of Microbial Isolates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was purified from 25 μl of each frozen isolate using Gentra Puregene Yeast/Bacteria kits, (Qiagen, Valencia, CA). We PCR amplified the intragenic spacer between 16 s and 23 s ribosomal subunits (ITS, 1256 nt)
[114 (link)] and portions of the nitrogenase α-subunit gene (NifD, 756 nt)
[115 (link)] as previously described
[66 (link)]. Amplification products were sequenced in both directions using an Applied Biosystems 96 capillary 3730xl DNA Analyzer (Foster City, CA) at the University of California, Berkeley, Sequencing Facility. Two to eight isolates from each plant were successfully amplified and sequenced, which ultimately yielded a sample of 84 sequenced isolates (Additional file
1 : Table S1).
Sequences were aligned using MAFFT
[116 (link)] with default parameters. Genotypes were identified in MacClade 4.05
[117 ]. Some portions of the ITS included indels and these regions were removed from the analysis.
[114 (link)] and portions of the nitrogenase α-subunit gene (NifD, 756 nt)
[115 (link)] as previously described
[66 (link)]. Amplification products were sequenced in both directions using an Applied Biosystems 96 capillary 3730xl DNA Analyzer (Foster City, CA) at the University of California, Berkeley, Sequencing Facility. Two to eight isolates from each plant were successfully amplified and sequenced, which ultimately yielded a sample of 84 sequenced isolates (Additional file
Sequences were aligned using MAFFT
[116 (link)] with default parameters. Genotypes were identified in MacClade 4.05
[117 ]. Some portions of the ITS included indels and these regions were removed from the analysis.
3
TLR Genetic Variants Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total genomic DNA was extracted from the peripheral blood, whole mouth saliva, and throat swabs samples using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instruction. Molecular typing of TLR2 (2029C/T, rs121917864; 2258G/A, rs5743708), TLR4 (896A/G, rs4986790) and TLR9 (− 1237T/C, rs5743836; − 1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) SNPs were performed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) as described elsewhere20 (link),41 (link). The results were confirmed by the sequencing of randomly selected samples of each TLR gene using the BigDye Terminator v3.1 Cycle Sequencing Kit and the 96-capillary 3730xl DNA Analyzer (Applied Biosystems).
4
Plasmid Construction and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids Construction of Bax, Bcl-2, p53, Cathepsin B, Caspase-3, Caspase-9 and EF-2 gene were performed as previously described (Mitupatum et al., 2015) . In brief, the PCR product of those amplified genes with the primer sets as shown in Table 1 was cloned into the TA cloning vector pTZ57R/T using the InsTAclone PCR Cloning Kit (Thermo Fisher Scientific, Pittsburgh, PA, USA). After transformation into competent cells (E. coli DH5α), the transformed cells were cultured and isolated for plasmid DNA by the QIAprep® Spin Miniprep Kit (QIAGEN, Hilden, Germany) before sequencing by Macrogen Inc. (Korea) with the 96-capillary 3730xl DNA Analyzer (Applied Biosystems®). Serial dilutions of plasmid DNA were performed before being added into the real-time PCR Master Mix. The gene expression analysis was carried out using the Exicycler™96 Bioneer (Bioneer Corporation, Daejoen, Korea). Ct value of each dilution was determined and then used to generate the standard curve against the logarithms of their own corresponding copy numbers. The curve was calculated by use of the equation previously described (Whelan et al., 2003) .
5
Fungal Genomic DNA Extraction and ITS Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA from the fungus was isolated from 7-d glucose-yeast medium mycelium using a Genomix DNA extraction kit (Talent, Italy) according to the manufacturer's instructions. The eluted DNA was stored at -20 ºC and used as a template for PCR amplifications. The ITS region was amplified by PCR using the primers and protocol described elsewhere (White et al. 1990 ). The PCR-amplified fragment was purified using the UltraCleanTM PCR Clean-Up TM kit (MoBio Laboratories Inc., USA) according to the manufacturer's instructions and was sent for direct sequencing to STABVIDA®. Automated sequencing of both strands was performed using the BigDye Terminator Kit from Applied Biosystems and the 96-capillary 3730xL DNA Analyzer from Applied Biosystems. The sequence was corrected using Chromas version 1.43 (Griffith University, Brisbane, Australia) and was analyzed and edited using Bioedit Sequence Alignment Editor version 7.0.9.0 (Hall 1999) . The sequence was deposited in the GenBank database under accession number KY174328.
6
Sanger Sequencing Workflow for Mutation Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five lines were randomly selected to verify the mutations that were identified bioinformatically with Sanger sequencing (Supporting Information, Table S1 ). Primers were designed using Primer3 (Rozen and Skaletsky 1999 ) and PCR products destined for sequencing were cleaned using a standard Exo-SAP protocol (Bell 2008 (link)), and sequenced with an ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Completed sequencing reactions were submitted to the Georgia Genomics Facility, and analyzed using an Applied Biosystems 3730xl 96-capillary DNA Analyzer.
7
RNA Isolation, cDNA Synthesis, and DNA Sequencing
8
Microsatellite Haplotyping of Disease Loci
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Using PCR and fluorescently labeled primers, we performed microsatellite sizing to determine the haplotypes. We used previously published primer sequences for the AR CAG disease expansion, the AR GGC repeat, and the DXS1194, DXS1111, DXS135, and DXS1125 microsatellites.9 (link),18 (link)- (link)23 (link) Primers for the microsatellite located in the DXS123 locus were created using Primer3web (primer3.ut.ee ). All primer sequences and optimized protocols are available on request. Forward primers for each primer pair were labeled with either 5-FAM or CAL Fluor Gold 540 (Biosearch Technologies, Hoddesdon, United Kingdom).
PCR was performed using 20 μL reaction volume and Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific), according to the manufacturer's protocols, for 40–42 cycles. The amplification of unique products was confirmed with agarose gel electrophoresis. The amplified products were analyzed in duplex (5-FAM/CAL Flour Gold 540) for cost-efficiency, using capillary electrophoresis (3730xl 96 capillary DNA Analyzer; Applied Biosystems, Waltham, MA). Fragment analysis was performed using Microsatellite Analysis Software (Applied Biosystems).
PCR was performed using 20 μL reaction volume and Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific), according to the manufacturer's protocols, for 40–42 cycles. The amplification of unique products was confirmed with agarose gel electrophoresis. The amplified products were analyzed in duplex (5-FAM/CAL Flour Gold 540) for cost-efficiency, using capillary electrophoresis (3730xl 96 capillary DNA Analyzer; Applied Biosystems, Waltham, MA). Fragment analysis was performed using Microsatellite Analysis Software (Applied Biosystems).
9
Sequencing of CRISPR-edited GmFAD2 genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The regions spanning two targets of GmFAD2 genes were amplified using Phusion® High-Fidelity DNA Polymerase (NEB, Ipswich, MA, USA) with different primer pairs for GmFAD2–1A or GmFAD2–1B (Additional file 1 : Table S1). The PCR products were separated by 1% agarose gel and, purified from gel and ligated to pGEM®-T Easy Vector Systems (Promega, Madison, WI, USA) for sequencing. The sequencing was performed utilizing a 3730xl 96-capillary DNA Analyzer with Applied Biosystems Big Dye Terminator cycle sequencing chemistry (ThermoFisher, Waltham, MA, US) at University of Missouri DNA Core. The sequences of transgenic and wild-type plants were aligned using online program MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/ ) to characterize the mutations induced by CRISPR/Cas9. The same sequencing approach was utilized to identify the inheritance of the mutations at T1 and T2 generations. In addition, Indel-specific primers were designed based on sequencing results and used to confirm the transmission of small deletions in progenies (Additional file 1 : Table S1).
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!