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Base Pairing: Pairing of purine and pyrimidine bases by Hydrogen bonding in double-stranded DNA or RNA.

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

1Department of Molecular Cell Biology, University of California, 2Howard Hughes Medical Institute, University of California, Berkeley, 3Innovative Genomics Institute, University of California, 4Biomedical Sciences Graduate Program, University of California, 5Department of Microbiology and Immunology, University of California, 6Diabetes Center, University of California, 7Chan Zuckerberg Biohub, 8Department of Medicine, University of California, 9UCSF Helen Diller Family Comprehensive Cancer Center, University of California, 10Department of Integrative Biology, University of California

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JoVE 57350


 JoVE In-Press

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

1Division of Hematology, Davidoff Cancer Center, Rabin Medical Center, 2The Center of Nanoscience and Nanotechnology, Tel Aviv University, 3Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, 4Department of Leukemia, The University of Texas MD Anderson Cancer Center

JoVE 53300


 Bioengineering

DNA Ligation Reactions

JoVE 5069

In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. This video provides an introduction to DNA ligation. The basic principle of ligation is described as well as a step-by-step procedure for setting up a generalized ligation reaction. Critical aspects of ligation reactions are discussed, such as how the length of a sticky end overhang affects the reaction temperature and how the ratio of DNA insert to vector should be tailored to prevent self-ligation. Molecular tools that assist with ligations like the Klenow Fragment and shrimp alkaline phosphatase (SAP) are mentioned, and applications , such as proximity ligations and the addition of linkers to fragments for sequencing are also presented.


 Basic Methods in Cellular and Molecular Biology

Zebrafish Microinjection Techniques

JoVE 5130

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage embryos. Using this technique, solutions containing genetic material or knockdown constructs are delivered into the blastomeres: the embryonic cells sitting atop the yolk of the newly fertilized egg. Delivery into the cytoplasm is achieved either through direct injection into the blastomere, or via natural cytoplasmic movements that occur after a solution is injected into the yolk. Successful genetic manipulations are usually followed by quantification of embryonic phenotypes in order to elucidate the genetic mechanisms of development. This video will provide an introduction to carrying out microinjections in zebrafish embryos. The discussion begins with a review of the essential tools for the technique, including the injection apparatus and the microinjector, which controls fluid movement with pressure pulses of air. Next, important preparatory steps are demonstrated, such as the pouring of agar plates to stabilize embryos during injection and calibration of the microinjection apparatus. The injection procedure is then presented along with tips on when and where injections should be performed. Finally, applications of the microinjection technique are discussed, including gene overexpression via mRNA inj


 Biology II

Gene Silencing with Morpholinos

JoVE 5326

Morpholino-mediated gene silencing is a common technique used to study roles of specific genes during development. Morpholinos inhibit gene expression by hybridizing to complementary mRNAs. Due to their unique chemistry, morpholinos are easy to produce and store, which makes them remarkably cost effective compared to other gene silencing methods.

This video reviews proper experimental design when using these oligonucleotides. Following that, an explanation of morpholino microinjection techniques in zebrafish and the analysis of resulting phenotypes will be discussed. Finally, we showcase examples of specific applications where morpholino technology is used to model developmental disorders or to study tissue regeneration.


 Developmental Biology

RNAi in C. elegans

JoVE 5105

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target gene. In the nematode, C. elegans, RNAi is particularly easy and effective because it can be delivered simply by feeding the worms bacteria that express double stranded RNA (dsRNA) that is complementary to a gene of interest. First, this video will introduce the concept of RNA interference and explain how it causes targeted gene knockdown. Then, we will demonstrate a protocol for using RNAi in C. elegans, which includes preparation of the bacteria and RNAi worm plates, culturing of the worms, and how to assess the effects of RNAi on the worms. RNAi is frequently used to perform reverse genetic screens in order to reveal which genes are important to carry out specific biological processes. Furthermore, automated reverse genetic screens allow for the efficient knockdown and analysis of a large collection of genes. Lastly, RNAi is often used to study the development of C. elegans. Since its discovery, scientists have used RNAi to make tremendous progress on the understanding of many biological phenomena.


 Biology I

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