Genomic DNA was extracted from PGCs using QIAMP Micro Kit (Qiagen) according to the manufacturer’s instruction. Specific primers for PCR amplification of sgRNA target sites were designed using primer3 software (http://primer3.ut.ee/ )90 (link),91 (link). Primers were designed to anneal outside the homology arms of HDR templates. List of primer sequences are listed in Supplementary Table S3 . All PCR amplifications were performed using 100 ng of genomic DNA and Q5® Hot Start High-Fidelity DNA polymerase (NEB) or Phusion® High-Fidelity DNA polymerase (NEB) according to the manufacturer’s protocol. Primer annealing temperatures were calculated using the online NEB Tm calculator (https://tmcalculator.neb.com ).
Qiamp micro kit
Manufactured by Qiagen
Sourced in United States
The QIAMP Micro Kit is a laboratory equipment product designed for the purification of DNA and RNA from small samples. It is a silica-membrane-based system that enables the efficient extraction and concentration of nucleic acids from various sample types, including tissues, cells, and body fluids.
Automatically generated - may contain errors
Lab products found in correlation
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Hiseq 2000 instrument, by Illumina (1 mentions)
Bioruptor ngs, by Diagenode (1 mentions)
Ampure xp pcr purification kit, by Beckman Coulter (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Wizard plus sv minipreps dna purification system, by Promega (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Qiamp dna stool mini kit, by Qiagen (1 mentions)
Matlab, by MathWorks (1 mentions)
Bigdye v3, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Exosap it, by US Biological (1 mentions)
5 protocols using qiamp micro kit
1
CRISPR Genomic DNA Extraction and PCR
2
Brown Bear DNA Extraction and Sequencing
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We extracted DNA from the Chichagof Island brown bears and the Swedish brown bear using the DNeasy Blood & Tissue Kit (Qiagen) and the QIAmp Micro Kit (Qiagen) according to the manufacturer's specifications. We physically sheared the DNA of the three new brown bear samples using a Diagenode Bioruptor NGS instrument. Extracts were transferred into 1.5-mL tubes and exposed to seven rounds of sonication, using the energy setting ‘HIGH’ and an ‘ON/OFF interval’ of 30/30 s. We then purified and concentrated the extracts using the Agencourt AMPure XP PCR purification kit, according to manufacturer's instructions, and eluted in 20 μL of 1xTE, with 0.05% Tween20.
We prepared indexed Illumina libraries using 15 μL of each extract following the protocol described by Meyer & Kircher (Meyer & Kircher 2010 (link)), with reaction volumes scaled to total volume of 40 μL. To verify final DNA concentration and the distribution of insert sizes, we ran each library on an Agilent 2100 Bioanalyzer. We then sequenced each bear on half of a lane of an Illumina HiSeq 2000 instrument using 150-base pair (bp) paired-end chemistry at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley.
We prepared indexed Illumina libraries using 15 μL of each extract following the protocol described by Meyer & Kircher (Meyer & Kircher 2010 (link)), with reaction volumes scaled to total volume of 40 μL. To verify final DNA concentration and the distribution of insert sizes, we ran each library on an Agilent 2100 Bioanalyzer. We then sequenced each bear on half of a lane of an Illumina HiSeq 2000 instrument using 150-base pair (bp) paired-end chemistry at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley.
3
Bisulfite Sequencing of Rat HSD11B2 Promoter
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Genomic DNA was extracted from placental samples using the QIAmp micro Kit (Qiagen, USA) following the manufacturer’s instructions, and treated with sodium bisulfite to convert non-methylated cytosine to uracil using EZ DNA Methylation Gold Kit (Zymo Research Corp., USA). The bisulfite reaction was performed on 0.5 μg DNA. PCR was done using specific primers (Assay ADS462; Rat HSD11B2 promoter ENSRNOT00000023130: −378 to −275 from start codon designed by EpigenDX, USA). The PCR products from bisulfite-treated genomic DNA were cloned into a pGEM-T Easy Vector (Promega, USA) following manufacturer’s instructions. Six to eight colonies from PCR cloning were selected and grown overnight in LB-Ampicillin medium at 37°C. The plasmid DNA was prepared using the Wizard plus SV Minipreps DNA purification system (Promega, USA), and sent to Macrogen (Korea) for sequentiation [29 ]. The PCR amplicon of 104 bp contained 10 CpG sites; however, we analyzed the first 5 CpG sites since they showed more than 90% matching identity with the promoter gene sequence (Rattus HSD11B2 cDNA; GenBank acc. NM_017081) [30 ] which was analyzed with BiQ Analyzer software (Fig. 1 ).
4
Gut Microbiome Diversity Analysis
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Genomic DNA was extracted from day-8 fecal samples ranging 5–20 mg, using Qiamp DNA Stool Mini kit (Qiagen), enzymatic lysis and mechanic disruption (30 (link)). Extra purification and concentration were performed following the cleaning protocol from Qiamp Micro kit (Qiagen). Fifty ng of DNA were amplified following the 16S Metagenomic Sequencing Library Illumina 15044223 B protocol (Illumina Inc, San Diego, CA, USA) and sequences were merged and processed using Pair-End read merger (PEAR v 0.9.6, Exelixis Lab, Heidelberg, Germany) and Cutadapt v1.8.1 (31 (link)).
To estimate the specific biodiversity, the Shannon–Wiener and CHAO1 indexes were calculated. In order to study the presence or absence of taxonomic ranks (family, genera and species) in the experimental groups, it was agreed that all groups displaying two or three animals (out of the three randomly analyzed in each group) with some bacterial proportion were computed as present, while the groups displaying one animal or none were computed as absent. Venn diagrams and heat maps based on log2 fold change with respect to 2′-FL were represented. Moreover, principal components analysis (PCA) was conducted to search for natural clustering of the samples with Matlab (MathWorks, Natick, MA, USA) using in-house scripts.
To estimate the specific biodiversity, the Shannon–Wiener and CHAO1 indexes were calculated. In order to study the presence or absence of taxonomic ranks (family, genera and species) in the experimental groups, it was agreed that all groups displaying two or three animals (out of the three randomly analyzed in each group) with some bacterial proportion were computed as present, while the groups displaying one animal or none were computed as absent. Venn diagrams and heat maps based on log2 fold change with respect to 2′-FL were represented. Moreover, principal components analysis (PCA) was conducted to search for natural clustering of the samples with Matlab (MathWorks, Natick, MA, USA) using in-house scripts.
5
Genomic DNA Extraction and Mitochondrial DNA Sequencing
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Genomic DNA was extracted from skin, muscle, and FTA cards using the DNeasy Blood and Tissue Kit (Qiagen). For tissue samples, we followed the manufacturer's animal tissue protocol. The FTA card DNA extraction required slight alterations (see electronic supplementary material). Some dart samples contained only hair and were extracted with the Qiamp Micro Kit (Qiagen) following the forensic case work protocol.
Mitochondrial DNA sequences DNA sequencing was accomplished by amplifying the hyper-variable region I (HVI) of the mitochondrial DNA control region using primers from Purdue et al. (2000) (see appendix for PCR conditions in electronic supplementary material). Amplification success was ascertained with 2 % agarose gels stained with ethidium bromide. Successful amplifications were purified using ExoSAP-IT (U.S. Biological, USA). Cycle sequencing reactions were performed in 10 ll reactions with 1 ll of purified PCR product, 1 lM primer, 0.25 ll BigDye v3.1, and 2.275 lL 59 sequencing buffer [Applied Biosystems (ABI), USA]. Sequencing was performed on an ABI 3130xl genetic analyzer.
Mitochondrial DNA sequences DNA sequencing was accomplished by amplifying the hyper-variable region I (HVI) of the mitochondrial DNA control region using primers from Purdue et al. (2000) (see appendix for PCR conditions in electronic supplementary material). Amplification success was ascertained with 2 % agarose gels stained with ethidium bromide. Successful amplifications were purified using ExoSAP-IT (U.S. Biological, USA). Cycle sequencing reactions were performed in 10 ll reactions with 1 ll of purified PCR product, 1 lM primer, 0.25 ll BigDye v3.1, and 2.275 lL 59 sequencing buffer [Applied Biosystems (ABI), USA]. Sequencing was performed on an ABI 3130xl genetic analyzer.
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