Total genomic DNA was extracted using a Plant Genomic DNA kit (TianGen, Beijing, China) following the manufacturer’s instructions. The detection of DNA degradation and contamination was done with 1% agarose gels and the DNA concentration was measured by Qubit® DNA Assay Kit in Qubit® 3.0 Fluorometer (Invitrogen, United States). The qualified DNA was digested by EcoRI, and P1 adapters were added at the ends of the digestion fragments. These fragments are subsequently pooled, randomly sheared, and size-selected. Then, the P2 adapter with a special “Y-type” structure was attached to the DNA fragments, ensuring that PCR only amplifies the sequences with two adapters at the same time. The quality of the libraries was checked by Qubit kit, Agilent2100, and Q-PCR. The qualified libraries were sequenced by the Illumina Hiseq platform to generate 150 bp paired-end reads (Miller et al., 2007 (link); Baird et al., 2008 (link)). At present, there is no complete genome of the Kadsura species, so the K. interior genome size estimated by flow cytometry (FCM) is used as a reference for sequencing the depth (Xu et al., 2021 (link)).
Qubit dna assay kit in qubit 3.0 fluorometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit® DNA Assay Kit is a sensitive and accurate fluorescence-based method for quantifying DNA in solution. When used with the Qubit® 3.0 Fluorometer, it provides a quick and reliable way to measure DNA concentration.
Automatically generated - may contain errors
Lab products found in correlation
Plant genomic dna kit, by Tiangen Biotech (1 mentions)
Nebnext enzymatic methyl seq kit, by New England Biolabs (1 mentions)
Sequencing platform, by Illumina (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Nebnext ultra dna library prep kit for, by Illumina (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanophotometer spectrophotometer, by Implen (1 mentions)
4 protocols using qubit dna assay kit in qubit 3.0 fluorometer
1
Kadsura genome sequencing protocol
2
Genomic DNA Extraction from R. japonica
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The seeds of R. japonica were collected from Lichuan City (Rj. LiChuan), Hubei Province of China; Shandong Province of China (Rj. ShanDong); and Beijing (Rj. BeiJing), China. All sample seedings were propagated in plastic pots containing a soil/vermiculite mixture (1:1) and the growth environments, as described in a previous study [25 (link)]. The fresh leaves of R. japonica were quickly frozen with liquid nitrogen immediately after picking and cleaning. Total whole genomic DNA was extracted with Super Plant Genomic DNA Kit (polysaccharide- and polyphenolics-rich) (Tiangen, Beijing, China), and purity and integrity of the extracted total genomic DNA were detected by 1% agarose gel electrophoresis, and the total concentration was estimated by Qubit DNA Assay Kit in Qubit 3.0 Fluorometer (Invitrogen, Waltham, MA, USA). The qualified samples were selected for subsequent experiments.
3
DNA Methylation Analysis of Diverse Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (about 1 × 109 cells) were Collected and used the QIAamp DNA Mini Kit (Qiagen, Germany) to extracted DNA according to the manufacturer’s instructions. Then DNA concentration was measured using Qubit® DNA Assay Kit in Qubit 3.0 Fluorometer (Life Technologies, CA, United States) and DNA quality was assessed by 1% agarose gel electrophoresis. Methylation libraries were constructed by using the NEBNext® Enzymatic Methyl-seq Kit (NEB) with 200 ng DNA as input amounts. And referred to the manufacturer’s protocol, we prepped libraries with fragments size of ∼400 bp for each sample (no biological replicates, a total of 3 libraries). High-throughput sequencing was performed using the Illumina sequencing platform, and the sequencing read length was 150 bp paired-end reads.
We got 3.3G of raw data in total. And bismark (Krueger and Andrews, 2011 (link)) was used to identify and extract methylation sites. The R package DSS (Wu et al., 2015 (link)) was used to identify differentially methylated regions of different components, using a threshold of P-value < 0.005.
We got 3.3G of raw data in total. And bismark (Krueger and Andrews, 2011 (link)) was used to identify and extract methylation sites. The R package DSS (Wu et al., 2015 (link)) was used to identify differentially methylated regions of different components, using a threshold of P-value < 0.005.
4
High-throughput DNA Sequencing of Recombinant Inbred Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The parents, Q9311B and WSSM, and the 1061 RILs were sequenced using the NGS platform. Genomic DNA for SNP genotyping was isolated from approximately 100 mg fresh leaf samples of 5-week-old seedlings for each of the 1061 RILs, Q9311B and WSSM using a modified cetyltrimethylammonium bromide method (Murray and Thompson 1980 (link)). DNA degradation and contamination were monitored on 0.8% agarose gels. DNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). DNA concentration was measured using Qubit® DNA Assay Kit in Qubit® 3.0 Fluorometer (Life Technologies, CA, USA).
The NEB Next® Ultra DNA Library Prep Kit for Illumina® (NEB, USA) was used to construct the libraries for sequencing as per the manufacturer’s instructions. DNA was fragmented into ~ 200 base pair pieces. The end of the DNA fragment was subjected to an end repair process that included the addition of a single “A” base, followed by ligation of the adapters. Products were purified and enriched by polymerase chain reaction (PCR) to amplify the library DNA. The final libraries were quantified using KAPA Library Quantification kit (KAPA Biosystems, South Africa) and an Agilent 2100 Bioanalyzer. Paired-end sequencing (2 × 150 basepair) was performed on an Illumina NovaSeq 6000 sequencer (Illumina, USA).
The NEB Next® Ultra DNA Library Prep Kit for Illumina® (NEB, USA) was used to construct the libraries for sequencing as per the manufacturer’s instructions. DNA was fragmented into ~ 200 base pair pieces. The end of the DNA fragment was subjected to an end repair process that included the addition of a single “A” base, followed by ligation of the adapters. Products were purified and enriched by polymerase chain reaction (PCR) to amplify the library DNA. The final libraries were quantified using KAPA Library Quantification kit (KAPA Biosystems, South Africa) and an Agilent 2100 Bioanalyzer. Paired-end sequencing (2 × 150 basepair) was performed on an Illumina NovaSeq 6000 sequencer (Illumina, USA).
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!