Like most other DNA sequencing core facilities, one of our continuing goals is to improve our sequence output without substantially adding to cost. To minimize sample-to-sample variability in template DNA concentration, we implemented the rolling circle amplification (RCA) procedure for preparing our DNA templates. In addition to saving time and reducing the number of steps in template DNA preparation, the RCA method has the potential to normalize the DNA concentration in samples that can be sequenced directly without additional purification. In the present study, we used RCA-generated templates to test a recently reported procedure that increased sequence quality by resuspending the sequenced products in low concentrations of agarose before capillary electrophoresis (CE) on a MegaBACE 1000 platform. Although we did not obtain the expected result using the specified procedure, a modification resulted in up to 60% increase in total sequence yield per sample plate. A combination of agarose and formamide-EDTA in the resuspension solution enabled us to generate long-read and high-quality sequences for more than 38,000 templates with minimal additional cost.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2279965 | PMC |
Publication Analysis
Top Keywords
Similar Publications
Targeted insertion of heterogenous DNA using Cas9-gRNA ribonucleoprotein-mediated gene editing in .
Bioengineered
December 2025
Department of BioMedical Bigdata (BK21) and Research Institute of Life Sciences, Gyeongsang National University, Jinju, Republic of Korea.
Gene editing is emerging as a powerful tool for introducing novel functionalities in mushrooms. While CRISPR/Cas9-induced double-strand breaks (DSBs) typically rely on non-homologous end joining (NHEJ) for gene disruption, precise insertion of heterologous DNA in mushrooms is less explored. Here, we evaluated the efficacy of inserting donor DNAs (8-1008 bp) with or without homologous arms at Cas9-gRNA RNP-induced DSBs.
View Article and Find Full Text PDFEstablishment of a human ovarian endometrioid carcinoma cell line by constitutive expression of cyclin-dependent kinase 4, cyclin D1 and telomerase reverse transcriptase.
Hum Cell
January 2025
Department of Tumor Pathology, Faculty of Medical Sciences, University of Fukui, 23-3 Matsuoka-Shimoaizuki, Eiheiji, Fukui, 910-1193, Japan.
Only a few human ovarian endometrioid carcinoma cell lines are currently available, partly due to the difficulty of establishing cell lines from low-grade cancers. Here, using a cell immortalization strategy consisting of i) inactivation of the p16-pRb pathway by constitutive expression of mutant cyclin-dependent kinase 4 (R24C) (CDK4) and cyclin D1, and ii) acquisition of telomerase reverse transcriptase (TERT) activity, we established a human ovarian endometrioid carcinoma cell line from a 46-year-old Japanese woman. That line, designated JFE-21, has proliferated continuously for over 6 months with a doubling time of ~ 55 h.
View Article and Find Full Text PDFsp. nov., isolated from the faecal sample of a zoo animal, .
Int J Syst Evol Microbiol
January 2025
Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, Republic of Korea.
Strain NoAH (=KACC 23135=JCM 35999), a novel Gram-negative, motile bacterium with a rod-shaped morphology, was isolated from the zoo animal faecal samples, specifically the long-tailed goral species . The novel bacterial strain grew optimally in a nutrient broth medium under the following conditions: 1-2% (w/v) NaCl, pH 7-8 and 30 °C. The strain NoAH exhibited high tolerance to NaCl, with the ability to tolerate up to 7% (w/v) NaCl.
View Article and Find Full Text PDFMetagenome-assembled genomes (MAGs) of the emerging pathogen from enrichment of coastal seawater, sediment, and algae at St. John's Island, Singapore.
Microbiol Resour Announc
January 2025
Singapore Centre for Environmental Life Sciences Engineering (SCELSE), National University of Singapore, , Singapore.
Coastal water, sediment, and algae samples were collected from St. John's Island, Singapore, and enriched in either broth or agar. Metagenomic sequencing was carried out on DNA from these enrichments and analyzed.
View Article and Find Full Text PDF<b>Background and Objective:</b> It is well documented that Whole Genome Sequencing (WGS) has recently used to explore new resistance patterns and track the dissemination of extensive and pan drug-resistant microbes in healthcare settings. This article explores the link between traumatic infections caused by road traffic accidents (RTAs) leading to coma and the development of chest infections caused by extensively drug-resistant (XDR) <i>Klebsiella pneumoniae</i> and <i>Pseudomonas aeruginosa</i>. <b>Materials and Methods:</b> The study was carried out from March to December 2022 which included a 45-year-old male patient admitted to the ICU of Al Ramadi Teaching Hospitals following a severe RTA that resulted in a TBI and subsequent coma.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!