Bio-protocol最新文献

{"title":"Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking.","authors":"Mengya Jia, Dongqin Ding, Xiaohua Liu, Haixing Li","doi":"10.21769/BioProtoc.5172","DOIUrl":"10.21769/BioProtoc.5172","url":null,"abstract":"<p><p>Genome walking is a popular molecular technique for accessing unknown flanking DNAs, which has been widely used in biology-related fields. Herein, a simple but accurate genome-walking protocol named partially overlapping primer (POP)-based PCR (POP-PCR) is described. This protocol exploits a POP set of three POPs to mediate genome walking. The three POPs have a 10 nt 3' overlap and 15 nt heterologous 5' regions. Therefore, a POP can partially anneal to the previous POP site only at a relatively low temperature (approximately 50 °C). In primary POP-PCR, the low-temperature (25 °C) cycle allows the primary POP to partially anneal to site(s) of an unknown flank and many sites of the genome, synthesizing many single-stranded DNAs. In the subsequent high-temperature (65 °C) cycle, the target single-stranded DNA is converted into double-stranded DNA by the sequence-specific primer, attributed to the presence of this primer complement, while non-target single-stranded DNA cannot become double-stranded because it lacks a binding site for both primers. As a result, only the target DNA is amplified in the remaining 65 °C cycles. In secondary or tertiary POP-PCR, the 50 °C cycle directs the POP to the previous POP site and synthesizes many single-stranded DNAs. However, as in the primary PCR, only the target DNA can be amplified in the subsequent 65 °C cycles. This POP-PCR protocol has many potential applications, such as screening microbes, identifying transgenic sites, or mining new genetic resources. Key features • This POP-PCR protocol, built upon the technique developed by Li et al. [1], is universal to genome walking of any species. • The established protocol relies on the 10 nt 3' overlap among a set of three POPs. • The first two rounds of POP-PCRs can generally give a positive walking outcome.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5172"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825308/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434565","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Flood Inoculation of <i>Fusarium eumartii</i> in Tomato Seedlings: Method for Evaluating the Infectivity of Pathogen Spores.","authors":"María Cecilia Terrile, Florencia Anabel Mesas, María Elisa Picco, María Florencia Salcedo, Andrea Yamila Mansilla","doi":"10.21769/BioProtoc.5198","DOIUrl":"10.21769/BioProtoc.5198","url":null,"abstract":"<p><p>The <i>Fusarium</i> genus includes various fungi of great significance in agriculture. <i>Fusarium solani</i> f. sp. <i>eumartii (F. eumartii</i>), traditionally known as a potato pathogen, has also been identified as a cause of disease in tomatoes. This protocol provides a detailed, efficient, and robust flood-inoculation method for assessing <i>F. eumartii</i> infection of young tomato seedlings grown on MS medium plates. It includes the evaluation of the lesion area and the quantification of the remaining fungal inoculum in tomato seedlings. In summary, the straightforwardness and efficiency of this bioassay make it a powerful quantitative tool for selecting fungicidal compounds or defense response inducers in tomato plants, offering a promising approach with significant potential for preventing fungal diseases in crops. Key features • Accelerated fungal infection method in tomato seedlings, shortening overall experimental time. • Allows simultaneous evaluation of fungal infectivity and quantitative remaining inoculum. • Easily adaptable for screening fungicides and defense inducers in various plant-pathogen interactions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5198"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825292/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of <i>Mycobacterium tuberculosis</i> and its Drug Resistance by Targeted Nanopore Sequencing Technology.","authors":"Chen Tang, Feng Xu, Xiaoqun Zheng, Guangxin Xiang","doi":"10.21769/BioProtoc.5170","DOIUrl":"10.21769/BioProtoc.5170","url":null,"abstract":"<p><p>Tuberculosis (TB) remains the leading cause of human mortality in infectious diseases. Drug-resistant TB, particularly multidrug-resistant TB and extensively drug-resistant TB, poses a pressing clinical and public health challenge. The main causative agents of TB are known as <i>Mycobacterium tuberculosis</i> (MTB), which exhibits a highly complex drug resistance profile. Traditional culture-based phenotypic drug susceptibility testing is time-consuming, and PCR-based assays are restricted to detecting known mutational hotspots. In this study, we present a protocol leveraging high-throughput nanopore sequencing technology in conjunction with multiplex PCR, termed targeted nanopore sequencing, for the identification of MTB and analysis of its drug resistance. Our method for MTB drug resistance assessment offers the benefits of being culture-free, efficient, high-throughput, and highly accurate, which could significantly aid in clinical patient management and the control of TB infections. Key features • Targeted nanopore sequencing detects 18 genes simultaneously linked to antibiotic resistance in MTB. • The method provides broad drug resistance profiles for 14 first- and second-line anti-TB drugs without bacterial culture. • The expedited turnaround time of the process is approximately 7.5 h with a detection limit of 10² bacteria/mL.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5170"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825305/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434529","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Profiling the Secretome of Glioblastoma Cells Under Histone Deacetylase Inhibition Using Mass Spectrometry.","authors":"Aline Menezes, Yara Martins, Fábio César Sousa Nogueira, Denise de Abreu Pereira, Katia Carneiro","doi":"10.21769/BioProtoc.5197","DOIUrl":"10.21769/BioProtoc.5197","url":null,"abstract":"<p><p>Glioblastoma (GBM) is the most aggressive brain tumor, and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. A label-free, mass spectrometry-based quantitative proteomics has been developed to identify and characterize proteins that are differentially expressed in GBM to gain a better understanding of the interactions and functions that lead to the pathological state focusing on the extracellular matrix (ECM). The main challenge in GBM research has been to identify novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. To better investigate the GBM secretome upon in vitro treatment with histone deacetylase inhibitor (iHDAC), we employed a high-throughput label-free methodology of protein identification and quantification based on mass spectrometry followed by in silico studies. Our analysis revealed significant changes in the ECM protein profile, particularly those associated with the angiogenic matrisome. Proteins such as decorin, ADAM10, ADAM12, and ADAM15 were differentially regulated upon in silico analysis. In contrast, key angiogenesis markers such as VEGF and ECM proteins like fibronectin and integrins did not display significant changes. These results suggest that iHDAC inhibitors may modulate or suppress tumor behavior growth by targeting ECM proteins' secretion rather than directly inhibiting angiogenesis. Key features • Analysis of the secretome of U87MG glioblastoma cells. • Studies of mass spectrometry designed to modulate GBM biology and behavior focused on histone deacetylase inhibitors (iHDAC). • Mass spectrometry was developed to identify and characterize proteins that are differentially expressed in GBM.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5197"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825294/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434476","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"FlashTag-mediated Labeling for Intraventricular Macrophages in the Embryonic Brain.","authors":"Hisa Asai, Mizuki Ono, Takaki Miyata, Yuki Hattori","doi":"10.21769/BioProtoc.5166","DOIUrl":"10.21769/BioProtoc.5166","url":null,"abstract":"<p><p>The fate mapping technique is essential for understanding how cells differentiate and organize into complex structures. Various methods are used in fate mapping, including dye injections, genetic labeling (e.g., Cre-lox recombination systems), and molecular markers to label cells and track their progeny. One such method, the FlashTag system, was originally developed to label neural progenitors. This technique involves injecting carboxyfluorescein diacetate succinimidyl ester (CFSE) into the lateral ventricles of mouse embryos, relying on the direct uptake of dye by cells. The injection of CFSE into the lateral ventricle allows for the pulse labeling of mitotic (M-phase) neural progenitors in the ventricular zone and their progeny throughout the brain. This approach enables us to trace the future locations and differentiation paths of neural progenitors. In our previous study, we adapted this method to selectively label central nervous system-associated macrophages (CAMs) in the lateral ventricle by using a lower concentration of CFSE compared to the original protocol. Microglia, the brain's immune cells, which play pivotal roles in both physiological and pathological contexts, begin colonizing the brain around embryonic day (E) 9.5 in mice, with their population expanding as development progresses. The modified FlashTag technique allowed us to trace the fate of intraventricular CAMs, revealing that certain populations of microglia are derived from these cells. The optimized approach offers deeper insights into the developmental trajectories of microglia. This protocol outlines the modified FlashTag method for labeling intraventricular CAMs, detailing the CFSE injection procedure, evaluation of CFSE dilution, and preparation of tissue for immunohistochemistry. Key features • This protocol builds upon the method developed by Govindan et al. and extends its application to intraventricular CAMs. • This protocol allows for the cell fate tracking of intraventricular CAMs within 24 h. • This protocol requires the technique of intraventricular injection of CFSE into embryonic brains.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5166"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769748/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054557","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Utilizing FRET-based Biosensors to Measure Cellular Phosphate Levels in Mycorrhizal Roots of <i>Brachypodium distachyon</i>.","authors":"Shiqi Zhang, Lucas Jurgensen, Maria J Harrison","doi":"10.21769/BioProtoc.5158","DOIUrl":"10.21769/BioProtoc.5158","url":null,"abstract":"<p><p>Arbuscular mycorrhizal (AM) fungi engage in symbiotic relationships with plants, influencing their phosphate (Pi) uptake pathways, metabolism, and root cell physiology. Despite the significant role of Pi, its distribution and response dynamics in mycorrhizal roots remain largely unexplored. While traditional techniques for Pi measurement have shed some light on this, real-time cellular-level monitoring has been a challenge. With the evolution of quantitative imaging with confocal microscopy, particularly the use of genetically encoded fluorescent sensors, live imaging of intracellular Pi concentrations is now achievable. Among these sensors, fluorescence resonance energy transfer (FRET)-based biosensors stand out for their accuracy. In this study, we employ the Pi-specific biosensor (cpFLIPPi-5.3m) targeted to the cytosol or plastids of <i>Brachypodium distachyon</i> plants, enabling us to monitor intracellular Pi dynamics during AM symbiosis. A complementary control sensor, cpFLIPPi-Null, is introduced to monitor non-Pi-specific changes. Leveraging a semi-automated ImageJ macro for sensitized FRET analysis, this method provides a precise and efficient way to determine relative intracellular Pi levels at the level of individual cells or organelles. Key features • This protocol describes the use of FRET biosensors for in vivo visualization of spatiotemporal phosphate levels with cellular and subcellular resolution in <i>Brachypodium distachyon</i>. • An optimized growth system can allow tracing of Pi transfer between AM fungi and host root. <b>This protocol is used in:</b> New Phytol (2022), DOI: 10.1111/nph.18081.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5158"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769715/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mouse-derived Synaptosomes Trypsin Cleavage Assay to Characterize Synaptic Protein Sub-localization.","authors":"Jasmeet Kaur Shergill, Domenico Azarnia Tehran","doi":"10.21769/BioProtoc.5164","DOIUrl":"10.21769/BioProtoc.5164","url":null,"abstract":"<p><p>Neurons communicate through neurotransmission at highly specialized junctions called synapses. Each neuron forms numerous synaptic connections, consisting of presynaptic and postsynaptic terminals. Upon the arrival of an action potential, neurotransmitters are released from the presynaptic site and diffuse across the synaptic cleft to bind specialized receptors at the postsynaptic terminal. This process is tightly regulated by several proteins at both presynaptic and postsynaptic sites. The localization, abundance, and function of these proteins are essential for productive neurotransmission and are often affected in neurological and neurodegenerative disorders. Here, we outline a method for purifying mouse synaptosomes and using limited tryptic digestion to assess the subcellular localization of synaptic proteins. During synaptosomes purification, presynaptic terminals reseal and are protected from proteolysis, while postsynaptic proteins remain susceptible to tryptic cleavage. These changes can easily be evaluated by western blot analysis. This approach offers a straightforward and reliable method to evaluate the subcellular localization of synaptic proteins based on their proteolytic sensitivity, providing valuable insights into synaptic physiology and pathology. Key features • Builds upon the method developed by Boyken et al. [1] and introduces the use of isolated mouse synaptosomes to assess synaptic protein sub-localization. • Limited tryptic digestion differentiates between presynaptic and postsynaptic proteins based on proteolytic sensitivity. • Requires standard biochemical reagents and western blotting equipment and can be completed in two/three days, including synaptosome purification and western blot analysis. Graphical overview <b>Overview of the synaptosomes trypsin cleavage assay.</b> This protocol describes the isolation of synaptosomes from mouse brain tissue, followed by limited trypsin digestion to assess the compartmental localization of synaptic proteins. Synaptosomes, which are isolated via differential centrifugation, consist of resealed presynaptic terminals that are protected from proteolysis, while the exposed postsynaptic compartments are accessible to trypsin and undergo proteolytic cleavage. Following digestion, samples are analyzed using SDS-PAGE and western blotting. Proteins of interest are probed using specific antibodies to determine whether they are presynaptic or postsynaptic. Presynaptic proteins (e.g., synaptophysin, SNAP-25) remain intact, while postsynaptic proteins (e.g., GluA1, GluN2A) are cleaved.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5164"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769747/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054510","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the <i>Tp53</i> Gene.","authors":"Srisathya Srinivasan, Hsin-Yi Henry Ho","doi":"10.21769/BioProtoc.5159","DOIUrl":"10.21769/BioProtoc.5159","url":null,"abstract":"<p><p>Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult. Previously described methods for MEF immortalization are often inconsistent or alter the physiological properties of the cells. Here, we describe an optimized method that overcomes these limitations. By using electroporation to deliver CRISPR constructs that target the <i>Tp53</i> gene, the method reliably generates immortalized MEFs (iMEFs) within three weeks. Importantly, iMEFs closely resemble the parent cell populations, and individual iMEFs can be cloned and expanded for subsequent genetic manipulation and characterization. We envision that this protocol can be adopted broadly to immortalize other mouse primary cell types. Key features • CRISPR-based knockout of the <i>Tp53</i> gene enables efficient immortalization of mouse embryonic fibroblasts (MEFs) in under three weeks. • Immortalization requires a Neon electroporator or a comparable system to transfect cells with the <i>Tp53</i> CRISPR constructs.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5159"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769752/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Using Protein Painting Mass Spectrometry to Define Ligand Receptor Interaction Sites for Acetylcholine Binding Protein.","authors":"Alexandru Graur, Natalie Erickson, Nadine Kabbani","doi":"10.21769/BioProtoc.5163","DOIUrl":"10.21769/BioProtoc.5163","url":null,"abstract":"<p><p>Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels expressed in nervous and non-nervous system tissue important for memory, movement, and sensory processes. The pharmacological targeting of nAChRs, using small molecules or peptides, is a promising approach for the development of compounds for the treatment of various human diseases including inflammatory and neurogenerative disorders such as Alzheimer's disease. Using the <i>Aplysia californica</i> acetylcholine binding protein (Ac-AChBP) as an established structural surrogate for human homopentameric α7 nAChRs, we describe an innovative protein painting mass spectrometry (MS) method that can be used to identify interaction sites for various ligands at the extracellular nAChR site. We describe how the use of small molecule dyes can be optimized to uncover contact sites for ligand-protein interactions based on MS detection. Protein painting MS has been recently shown to be an effective tool for the identification of residues within Ac-AChBP involved in the binding of know ligands such as α-bungarotoxin. This strategy can be used with computational structural modeling to identify binding regions involved in drug targeting at the nAChR. Key features • Identify binding ligands of nicotinic receptors based on similarity with the acetylcholine binding protein. • Can be adapted to test various ligands and binding conditions. • Mass spectrometry identification of specific amino acid residues that contribute to protein binding. • Can be effectively coupled to structural modeling analysis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5163"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769746/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054576","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cloning a Chloroplast Genome in <i>Saccharomyces cerevisiae</i> and <i>Escherichia coli</i>.","authors":"Emma Jane Lougheed Walker, Bogumil Jacek Karas","doi":"10.21769/BioProtoc.5162","DOIUrl":"10.21769/BioProtoc.5162","url":null,"abstract":"<p><p>Chloroplast genomes present an alternative strategy for large-scale engineering of photosynthetic eukaryotes. Prior to our work, the chloroplast genomes of <i>Chlamydomonas reinhardtii</i> (204 kb) and <i>Zea mays</i> (140 kb) had been cloned using bacterial and yeast artificial chromosome (BAC/YAC) libraries, respectively. These methods lack design flexibility as they are reliant upon the random capture of genomic fragments during BAC/YAC library creation; additionally, both demonstrated a low efficiency (≤ 10%) for correct assembly of the genome in yeast. With this in mind, we sought to create a highly flexible and efficient approach for assembling the 117 kb chloroplast genome of <i>Phaeodactylum tricornutum</i>, a photosynthetic marine diatom. Our original article demonstrated a PCR-based approach for cloning the <i>P. tricornutum</i> chloroplast genome that had 90%-100% efficiency when screening as few as 10 yeast colonies following assembly. In this article, we will discuss this approach in greater depth as we believe this technique could be extrapolated to other species, particularly those with a similar chloroplast genome size and architecture. Key features • Large fragments of the chloroplast genome can be readily amplified through PCR from total algal DNA isolate. • Assembly protocol can be completed within a day, and yeast colonies harboring chloroplast genomes can be obtained in as few as 4-5 days. • Cloned genomes isolated from yeast transformants can be moved to <i>Escherichia coli</i> through electroporation.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5162"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769754/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054550","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}