HeLa-S3, IMR-90, and K562 gDNAs were purchased from MilliporeSigma (catalog #87110901, 85020204, 89121407). GM12878 gDNA was purchased from the Coriell Institute for Medical Research (#NA12878). Methylated lambda bacteriophage gDNA, from strain cI857 Sam7 grown in E. coli strain W3110, was purchased from Thermo Fisher Scientific (#SD0011). Unmethylated lambda gDNA, from the same virus strain grown in dam−dcm−E. coli strain GM2163, was purchased from Thermo Fisher Scientific (#SD0021). All gDNA samples were requantified with a Qubit 1X dsDNA High-Sensitivity kit (#Q33230; Thermo Fisher Scientific) and diluted according to this measurement in nuclease-free 1X TE buffer with 0.05% wt/vol Tween 20. All experiments used 60 ng gDNA except those comparing lower inputs, for which gDNA was serially diluted and technical replicates were taken from the same dilution.
Qubit 1x dsdna high sensitivity kit
Manufactured by Thermo Fisher Scientific
Sourced in Singapore
The Qubit 1X dsDNA High-Sensitivity kit is a fluorometric quantitation method for measuring double-stranded DNA (dsDNA) concentrations. It uses a fluorescent dye that specifically binds to dsDNA, allowing for accurate and sensitive measurement of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Gm12878 gdna, by Coriell Institute for Medical Research (2 mentions)
Methylated lambda bacteriophage gdna, by Thermo Fisher Scientific (2 mentions)
Unmethylated lambda gdna, by Thermo Fisher Scientific (2 mentions)
Gm2163, by Thermo Fisher Scientific (2 mentions)
Hela s3, by Merck Group (2 mentions)
Imr90, by Merck Group (2 mentions)
Nextseq 550 platform, by Illumina (2 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
High sensitivity dna chip, by Agilent Technologies (2 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Innuprep plant dna kit, by Analytik Jena (1 mentions)
Dnazol, by Molecular Research Center (1 mentions)
Rnase if, by New England Biolabs (1 mentions)
Dna clean concentrator 25 kit, by Zymo Research (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Rneasy total rna kit, by Qiagen (1 mentions)
Qubit flex fluorometer, by Thermo Fisher Scientific (1 mentions)
6 protocols using qubit 1x dsdna high sensitivity kit
1
Comparative Genomic DNA Profiling
2
Benchmarking DNA Quantification Protocols
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HeLa-S3, IMR-90, and K562 gDNAs were purchased from MilliporeSigma (catalog #87110901, 85020204, 89121407). GM12878 gDNA was purchased from the Coriell Institute for Medical Research (#NA12878). Methylated lambda bacteriophage gDNA, from strain cI857 Sam7 grown in E. coli strain W3110, was purchased from Thermo Fisher Scientific (#SD0011). Unmethylated lambda gDNA, from the same virus strain grown in dam−dcm−E. coli strain GM2163, was purchased from Thermo Fisher Scientific (#SD0021). All gDNA samples were requantified with a Qubit 1X dsDNA High Sensitivity kit (Thermo Fisher Scientific #Q33230) and diluted according to this measurement in nuclease-free 1X TE buffer with 0.05% w/v Tween 20. All experiments used 60ng gDNA except those comparing lower inputs, for which gDNA was serially diluted and technical replicates were taken from the same dilution.
3
Plant DNA Extraction and Amplicon Sequencing
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We used the innuPREP Plant DNA Kit (Analytik Jena) to extract plant genomic DNA. The targeted sequences were amplified with specific primers (Additional file 1 : Table S5), and the amplicons were purified with the GeneJET Gel Extraction Kit (Thermo Fisher Scientific) then quantified using a Qubit™ 1X dsDNA High Sensitivity Kit (Thermo Fisher Scientific). Oligos used in this study are listed in Additional file 1 : Table S5. Equal amounts of PCR products were pooled and sequenced (GENEWIZ, AMPLICON-EZ). Amplicon deep sequencing was performed three times for each target location using genomic DNA isolated from three different protoplast transformation experiments. The target sites in the sequenced reads were analyzed for mutations using CRISPResso2 (Additional file 1 : Table S6) [49 (link)].
4
DNA Extraction from Frozen Samples
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DNA was extracted with DNAzol (Molecular Research Center, Cincinnati, OH; DN127) as follows: Frozen samples were pulverized with a plastic pestle, then mixed with 200 μL of DNAzol and incubated at room temperature for 10 minutes. A total of 100 μL of 100% EtOH was added to each sample, mixed thoroughly and centrifuged for 5 minutes at 15,000 xg. Supernatant was discarded, and the pellet was washed with a mixture of 70% DNAzol and 30% EtOH, followed by a second wash with 70% EtOH. After removing the supernatant, the pellet was air dried for 1 minute, then resuspended in 30 μL TE buffer (10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA). DNA was treated with RNAse If (New England Biolabs, Ipswich, MA; M0243) for 10 minutes at room temperature, purified using the DNA Clean & Concentrator-25 Kit (Zymo Research, Irvine, MA; D4033) according to the manufacturer’s protocol, and eluted in 30 μL TE buffer. DNA concentration was measured with a Qubit 4.0 using the Qubit 1x dsDNA High Sensitivity kit (Thermo Fisher Scientific, Waltham, MA; Q33230).
5
Small RNA Sequencing Library Preparation
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Small RNA library preparation for sequencing was adapted from Hagemann-Jensen et al. (2018) (link). cDNA libraries were prepared on a 96-well-plate using 3 µl of input RNA from each sample. A thermal cycler from BIOER Life Touch (Techtum, China) was used in all steps. After the second PCR, the cDNA libraries were purified individually with AMPure XP beads (Beckman Coulter Inc., USA) in a 1:1 bead to sample ratio. The quality of the purified cDNA product was checked on a High Sensitivity DNA chip (Agilent Technologies, USA) using an Agilent 2100 Bioanalyzer System (Agilent Technologies, USA) and the quantity was measured with a Qubit 4 Fluorometer (Invitrogen, Singapore) using the Qubit 1X dsDNA High Sensitivity Kit (Invitrogen, Oregon, USA). Samples were indexed using customized barcodes (IDT Technologies, USA). From each library, 5 ng of cDNA was pooled and small RNA sequencing was performed on an Illumina NextSeq 550 platform at the SciLifeLab, National Genomics Infrastructure, Karolinska Institutet.
6
Sensitive Small RNA Sequencing Protocol
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Library preparation: Total RNA was extracted from tissue stored in RNA later using RNeasy total RNA Kit (Qiagen, Hilden, Germany) as per the manufacturer’s protocol. cDNA libraries for small RNA sequencing were constructed according to a highly sensitive small RNA sequencing protocol (26 (link)) with 1ng of total RNA as starting material. Each sample was indexed using customized barcodes (IDT Technologies, Germany), and the 10ng library from each is pooled and sequenced. A thermal cycler from BIOER Life Touch (Techtum, China) was used in all the steps. The final cDNA libraries quality was checked on a High Sensitivity DNA chip (Agilent Technologies, USA) using Agilent 2100 Bioanalyzer System (Agilent Technologies, USA). DNA quantity was determined with Qubit Flex Fluorometer (Invitrogen, Singapore) using the Qubit 1X dsDNA High Sensitivity Kit (Invitrogen, Oregon, USA). Sequencing was performed on the Illumina NextSeq 550 platform with 1x75 basepairs, and single-end reads at the Bioinformatics and Expression Analysis core facility at Karolinska University Hospital, Sweden.
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