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Molecular Biology

protocols
Protocol

E. coli Expression Experimental Protocol

Experiment Tags: E. coli Expression, Cloning, plasmid preparation

Experiment Summary: Recombinant protein expression in Escherichia coli (E. coli) is widely used in molecular biology and biotechnology due to its ease, cost-effectiveness, and rapid growth. This E. coli expression protocol outlines a standardized workflow for cloning, transformation, protein expression, and purification in the E. coli system, ensuring reproducibility and efficiency.

protocols
Protocol

Plasmid Purification And Troubleshooting

Experiment Tags: Plasmid purification, Plasmid purification

Experiment Summary: Plasmids are small circular deoxyribonucleic acids (DNA) outside the chromosomes that replicate independently of chromosomal DNA. Although budding yeast and fission yeast can retain plasmids, the host of plasmids is almost always bacteria. This small circular DNA is widely used as a DNA vector in the fields of molecular biology, biochemistry, biotechnology, cell biology, etc.

protocols
Protocol

Bacterial Transformation Protocol

Experiment Tags: Bacterial Transformation, Bacterial Transformation Protocol, transformation, bacterial cells

Experiment Summary: Bacterial transformation is a fundamental process in molecular biology, enabling the introduction of foreign DNA into bacterial cells. Primarily used for the amplification and storage of plasmids, bacterial transformation is critical for cloning experiments.

protocols
Protocol

CRISPR/Cas9-Mediated Gene Editing in C. elegans

Experiment Tags: CRISPR-Cas9, Gene Editing, C. elegans, sgRNA, Plasmid Construction Homologous Recombination, Genetic Screening.

Experiment Summary: This protocol outlines the process for performing CRISPR/Cas9-mediated genome modifications in C. elegans, including the design of specific sgRNA sequences, construction of Cas9-sgRNA plasmids, and preparation of injection mixtures.

protocols
Protocol

Rapid and Efficient Gene Editing in Human Pluripotent Stem Cells via FLPe Recombinase-Mediated Cassette Exchange at the AAVS1 Locus

Experiment Tags: Gene Editing, FLPe Recombinase, Cassette Exchange, AAVS1 Locus, Human Pluripotent Stem Cells

Experiment Summary: The protocol presents a unique approach to FLPe recombinase-mediated cassette exchange (RMCE) gene editing at the AAVS1 locus in human pluripotent stem cells (hPSCs) that is both rapid and efficient. This method makes it easier to generate highly reliable isogenic lines, which in turn makes transgenesis studies in hPSCs easier to conduct.

protocols
Protocol

Protocol for CRISPR/Cas9-mediated Knockout in Difficult-to-Transfect Cells

Experiment Tags: CRISPR/Cas9, Lentivirus, Gene Knockout, Cell Line Development

Experiment Summary: The protocol utilizes lentiviral vectors and a control system to regulate Cas9 activity, enabling the development of knockout cell lines. The development of a human muscle cell line that targets the gene serves as evidence of the protocol's effectiveness.

protocols
Protocol

Protocol for rAAVrh.74-SaCas9/gRNA Vector-Mediated Postnatal Cardiac Gene Editing in mdx/utrophin+/- Mice

Experiment Tags: CRISPR, Genome Engineering, Cardiac Gene Editing, rAAV Delivery, Dystrophin Restoration

Experiment Summary: This protocol describes how to use the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system to perform postnatal cardiac gene editing in mdx/utrophin+/- mice. Specifically, the protocol uses the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and guide RNAs (gRNAs) delivered via recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) to carry out the gene editing. Restoring dystrophin expression in these mice's cardiac muscles is the main goal in hopes of developing a treatment for Duchenne muscular dystrophy (DMD).

protocols
Protocol

Recombinase-Mediated Cassette Exchange for Structure-Function Studies in Mouse Embryonic Stem Cells

Experiment Tags: RMCE, Mouse Embryonic Stem Cells, Structure-Function Studies, Recombinase-Mediated Cassette Exchange, Gene Targeting, ROSALUC Mice, R26-iPSC Mice, pRMCE-DV1, Cre-Excised pRMCE-DV1, pCAG-FlpE-IRES-Puro-pA, Gateway pDONR221 Vector.

Experiment Summary: This protocol describes how to employ recombinase-mediated cassette exchange (RMCE) to perform structure-function research in mouse embryonic stem cells (mESCs). The process entails separating KO mESCs that are compatible with RMCE, creating a targeting vector that is compatible with RMCE, and using RMCE to target rescue constructs to the ROSA26 locus using mESCs.

protocols
Protocol

Optimization of CRISPR-Cas9 Gene Editing in Brown Adipose Tissue Using Lentivirus and AAV Delivery Systems

Experiment Tags: CRISPR-Cas9, Gene Editing, Lentivirus, AAV, Brown Adipose Tissue, Mouse Model

Experiment Summary: This Protocol optimizes CRISPR-Cas9 gene editing in brown adipose tissue (BAT) using lentivirus and AAV systems. Ucp1-Cre/Cas9 mice are used in in vivo research after screening effective sgRNAs in cultured cells and cloning them into AAV vectors. Western blot analysis is used to validate the effectiveness of gene deletion. By using this approach, the biology of BAT and its potential for treatment in metabolic diseases are better understood.

protocols
Protocol

CRISPR-based Gene Editing Protocol for Jurkat T Cells

Experiment Tags: CRISPR, Gene Editing, Jurkat T Cells, Lentivirus, dCas9, gRNA, IFNG-AS1

Experiment Summary: This protocol outlines a comprehensive procedure for CRISPR-based gene editing in Jurkat T cells using lentiviral vectors encoding dCas9 and guide RNAs (gRNAs). The experiment includes vector design and generation, virus generation and particle count, dCas9-VP64 transduction, selection, clone creation, and screening.

* For research use only. Not intended for any clinical use.
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