To validate the methylation state of the methylation peak based on MeDIP-Seq data, bisulfite genomic sequencing PCR (BSP) was performed (Sigma, USA). We designed a bisulfite-modified PCR primer pair from horse genomic sequences for the promoter regions of three genes using the MethPrimer program (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi ). The primer information is listed in Supplementary Table 2 . PCR amplification was performed in each DNA sample using the following conditions: 94°C for 4 min, followed by 35 cycles of 94°C for 40 s, annealing at 55°C for 40 s, elongation at 72°C for 1 min, and final elongation at 72°C for 7 min using a Mastercycler® pro S thermal cycler (Eppendorf, Germany). The PCR products were separated on a 1.5% agarose gel, purified with a Labopass gel extraction kit (Cosmogenetech, Korea), and cloned into the pGEM-T Easy vector (Promega). Ten individual clones were sequenced by an automated 3730 DNA analyzer (Applied Biosystems, USA).
Automated 3730 dna analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Automated 3730 DNA Analyzer is a high-throughput capillary electrophoresis instrument designed for DNA sequencing applications. It utilizes a 48-capillary array to simultaneously analyze multiple DNA samples. The system is capable of generating DNA sequence data through fluorescence-based detection.
Automatically generated - may contain errors
Lab products found in correlation
Genemapper software version 4, by Thermo Fisher Scientific (2 mentions)
Pgem t easy vector, by Promega (1 mentions)
Labopass gel extraction kit, by Cosmogenetech (1 mentions)
Mastercycler pro s thermal cycler, by Eppendorf (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Tsq quantum mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Accela autosampler, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
P131 ccm, by MRC-Holland (1 mentions)
Salsa mlpa kits p130, by MRC-Holland (1 mentions)
Ampflstr identifiler kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using automated 3730 dna analyzer
1
Bisulfite Genomic Sequencing for DNA Methylation
2
Genotyping of IL-28 Polymorphisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNAs were isolated from 0.5 mL whole blood using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). IL-28 rs12979860, rs8099917, rs10853728, rs12980275, rs4803219, rs4803223, rs8105790 and rs28416813 were amplified by PCR. The PCR protocol involved initial denaturation at 95°C for 10 min, 35 cycles of denaturation for 30 s at 95°C, annealing of primers for 30 s at 55°C and extension for 40 s at 72°C, followed by final extension at 72°C for 10 min. The amplified DNA fragments were separated on a 2% agarose gel, and purified with the QIAquick gel extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions Nucleotide sequences were determined by Sanger sequencing using the Applied Biosystems Automated 3730 DNA Analyzer.
3
Folate, Choline, and Metabolite Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum and red blood cell folate concentrations were determined by microbiological assay using Lactobacillus rhamnosus55 (link),56 (link). The inter- and intra-assay coefficients of variation were 7.7% and 6.7%, respectively, for serum folate and 5.1% and 3.3%, respectively for RBC folate. Plasma choline, betaine, dimethylglycine (DMG), and trimethylamine N-oxide (TMAO) concentrations were determined using LC-MS/MS stable isotope dilution methods as described by Yan et al.57 (link) using a TSQ Quantum mass spectrometer (Thermo) with refrigerated Accela autosampler (Thermo) and Accela pump with degrasser (Thermo). MTHFR 677 C → T (rs18001133) genotype was determined after purifying PCR products (QUIquick PCR Purification kit) and sequencing DNA templates with an Applied Biosystems Automated 3730 DNA analyzer (Applied Biosystems) by the Georgia Genomics Facility (Athens, GA).
4
DNA Purification and Sequencing Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
qPCR products from amplification of DNA from the EW1 and O-BH9-A1 wells were purified using the QIAquick PCR Purification Kit (Qiagen). DNA sequencing was performed using an Applied Biosystems Automated 3730 DNA Analyzer at the Cornell University Life Sciences Core Laboratories Center. Trimmed sequences were analyzed using a NCBI- BLASTn query against the nr database.
5
Multiplex Ligation-Dependent Probe Amplification for CCM Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Multiplex ligation-dependent probe amplification (MLPA) was performed on patients who were negative for direct sequencing analysis for mutation in KRIT1/CCM1, MGC4607/CCM2 and PDCD10/CCM3 by using two MLPA kits (SALSA MLPA Kits P130 & P131 CCM, MRCHolland). The P130 probe mix contains probes for part of KRIT1/CCM1 exons and for all MGC4607/CCM2 exons. The P131 probe mix contains probes for the remaining KRIT1/CCM1 exons and for all exons of PDCD10/CCM3 gene. MLPA was performed according to the protocol supplied, by use of 100 ng of DNA sample per reaction, using FAM labelled primers. Samples were run on a 3730 DNA automated analyzer (Applied Biosystems), and data were analyzed with the GeneMapper software version 4.0 (Applied Biosystems) to size the PCR products and to obtain peak areas.
For the visual inspection, peak heights were compared between the samples and the controls, to find any alteration in relative peak heights within the test sample. For the normalized peak area calculations, each peak area was normalized by dividing the individual peak area by the total peak area of all peaks for that sample. See Penco et al., 2009 for details [24] (link).
For the visual inspection, peak heights were compared between the samples and the controls, to find any alteration in relative peak heights within the test sample. For the normalized peak area calculations, each peak area was normalized by dividing the individual peak area by the total peak area of all peaks for that sample. See Penco et al., 2009 for details [24] (link).
6
STR Multiplex Assay with Identifiler Kit
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
STR (Short Tandem Repeat) multiplex assay was performed by using the AmpFlSTR Identifiler Kit (Applied Biosystems) according to the manufacturer's instructions. The kit has been designed to amplify 15 tetranucleotide repeat loci and the amelogenin gender-determining marker in a single PCR amplification; a five-dye fluorescent system was used for automated DNA fragment analysis.
Samples were run on a 3730 DNA automated analyzer (Applied Biosystems), and data were analyzed with the Gene Mapper software version 4.0 (Applied Biosystems); allele peaks were interpreted when the peak heights were greater than or equal to 50 relative fluorescence units.
Samples were run on a 3730 DNA automated analyzer (Applied Biosystems), and data were analyzed with the Gene Mapper software version 4.0 (Applied Biosystems); allele peaks were interpreted when the peak heights were greater than or equal to 50 relative fluorescence units.
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!