Presented here is a complete molecular cloning protocol consisting of a number of separate but interconnected methods such as preparation of E. coli competent cells; in vitro DNA digestion and ligation; PCR; DNA agarose gel electrophoresis and gel extraction; and screening transformants by colony PCR, analytical restriction digests, and sequencing. The method is described in a lot of details so that it can be easily followed by those with very little relevant knowledge and skills. It also contains many tips that even experienced researchers may find useful.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-62703-565-1_2 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Cloning, purification and characterization of a novel thermostable recombinant tannase from Galactobacillus timonensis.
Enzyme Microb Technol
December 2024
State Key Laboratory of Food Science and Resources, School of Food Science and Technology, Nanchang University, Nanchang 330047, China.
The exorbitant production costs associated with natural tannases pose a significant challenge to their widespread industrial utilization. Microbial expression systems provide a cost-effective method for enzyme production. In this study, a putative gene encoding the subtype B tannase (Gt-Tan) was cloned from Galactobacillus timonensis and expressed heterologously in Escherichia coli BL21 (DE3) cells.
View Article and Find Full Text PDFPinus radiata cDNA Library for the Screening of Phytophthora Effector Protein Targets in Yeast.
Methods Mol Biol
December 2024
School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.
Yeast two-hybrid library screening enables the discovery of novel protein-protein interactions. Identifying cytosolic host proteins targeted by host-translocated Phytophthora effector proteins relies on the mRNA amount, quality, and composition used to prepare the yeast two-hybrid cDNA library. Here we describe the steps required for the preparation of a Pinus radiata cDNA library optimized for Phytophthora effector target screening in yeast.
View Article and Find Full Text PDFMolecular Characterization, Expression Analysis, and CRISPR/Cas9 Mediated Gene Disruption of Myogenic Regulatory Factor 4 (MRF4) in Nile Tilapia.
Curr Issues Mol Biol
December 2024
Department of Fisheries Science, Chonnam National University, Yeosu 59626, Republic of Korea.
Myogenic regulator factors (MRFs) are essential for skeletal muscle development in vertebrates, including fish. This study aimed to characterize the role of () in muscle development in Nile tilapia by cloning from muscle tissues. To explore the function of , CRISPR/Cas9 gene editing was employed.
View Article and Find Full Text PDFIdentification and characterization of a novel chromosome-encoded aminoglycoside -nucleotidyltransferase gene, in sp. TYF-12 isolated from the marine fish intestine.
Front Microbiol
December 2024
Department of Laboratory Sciences, The People's Hospital of Yuhuan, Yuhuan, China.
Background: The mechanisms underlying the resistance of the genus to aminoglycosides are complex, which poses a challenge for the efficient treatment of infectious diseases caused by these pathogens. To help clinicians treat infections more effectively, a more comprehensive understanding of antibiotic resistance mechanisms is urgently needed.
Methods: Plates were streaked to isolate bacteria from the intestinal contents of fish.
In silico design and assessment of a multi-epitope peptide vaccine against multidrug-resistant .
In Silico Pharmacol
December 2024
Department of Microbiology, Panjab University, Chandigarh, 160014 India.
Unlabelled: , an opportunistic and notorious nosocomial pathogen, is responsible for many infections affecting soft tissues, skin, lungs, bloodstream, and urinary tract, accounting for more than 722,000 cases annually. Despite the numerous advancements in therapeutic options, no approved vaccine is currently available for this particular bacterium. Consequently, this study focused on creating a rational vaccine design using bioinformatics tools.
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!