SCIENCE EDUCATION > Basic Biology

Basic Methods in Cellular and Molecular Biology

This collection demonstrates how to execute basic techniques commonly used in cellular and molecular biology.

  • Basic Methods in Cellular and Molecular Biology

    10:18
    Using a Hemacytometer to Count Cells

    Many biomedical experiments require manipulation of a known quantity of cells, in order to achieve accurate, reproducible, and statistically-relevant data. Therefore, learning how to count cells is a particularly essential technique for any successful biomedical scientist. The most common way to count cells is by using a hemacytometer - an instrument that bears two laser-etched grids, which aid in the enumeration of an aliquot cells under a simple light microscope. This data can then be used to extrapolate the number of cells in experimental sample. This video will show how to: adjust the experimental sample concentration so that you are not trying to count too many – or too few – cells; how to use a hemacytometer to count a small (~10 μl) aliquot of cells; how to determine which quadrant of the hemacytometer laser grid to use for counting; how to calculate the total number of cells in your experimental sample, depending upon which quadrant was used; and how to determine the viability of your experimental cell population using trypan blue exclusion. Further, various experimental situations for which reliable and accurate determination of cell numbers is necessary, including an example using an automated cell counter, are also discussed.

  • Basic Methods in Cellular and Molecular Biology

    09:44
    Restriction Enzyme Digests

    Restriction enzymes or endonucleases recognize and cut DNA at a specific sequence. These enzymes occur naturally in bacteria as a defense against bacteriophages - viruses that infect bacteria. Bacterial restriction enzymes cut the invading bacteriophage DNA while leaving the bacterial genomic DNA unharmed due to addition of methyl groups.

    This video explains the basic principles of restriction enzymes including: how restriction enzymes are named and the types of recognition sites and overhangs that exist. Also provided is a step-by-step generalized procedure for how to set up a restriction digest including the necessary components, the order in which the mixture should be assembled, and the typical incubation temperature and time. The importance of inactivating restriction enzymes to prevent star activity is mentioned. Tips for performing multiple enzymes digests and using controls in digestion reactions are also provided.

  • Basic Methods in Cellular and Molecular Biology

    07:33
    DNA Ligation Reactions

    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

    07:27
    An Introduction to Transfection

    Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for transfected RNA is the cytoplasm, DNA must be transported to the nucleus for effective transfection. There, the DNA can be transiently expressed for a short period of time, or become incorporated into the genomic DNA, where the change is passed on from cell to cell as it divides. This video describes the basics behind chemical mediated transfections and introduces some of the most commonly-used reagents, including charged lipids, polymers, and calcium phosphate. Each step is described from the preparation of cells for transfection through analysis of transfection efficiency. Additionally, the applications section of this video-article describes the use of electroporation and a biolistic transfection as alternative methods for introducing nucleic acid into mammalian cells. It also describes an advanced use of transfection where co-transfection of interfering RNA and DNA are introduced as a way to down-regulate a naturally occurring protein while at the same time producing a mutant variant of it within the same cell.

  • Basic Methods in Cellular and Molecular Biology

    08:47
    The Western Blot

    Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry. This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. The broad applications of this technique are described through several examples including the detection of protein-prot

  • Basic Methods in Cellular and Molecular Biology

    06:29
    Gel Purification

    Gel purification is used to recover DNA fragments after electrophoretic separation. DNA recovery from an agarose gel includes three basic steps: binding, washing and eluting from a silica column. DNA is believed to bind to silica in the presence of high salt via a salt bridge. Following binding, DNA is washed of impurities and eluted under low salt conditions disrupting this interaction.

    This video goes through a step-by-step, generalized procedure for cutting out a band from the gel, gel solubilization, purification through binding to a silica column, and elution of purified DNA. In addition, the presentation discusses several tips for ensuring successful gel purification, including the importance of running an agarose gel with a marker or ladder that has DNA of known sizes.

  • Basic Methods in Cellular and Molecular Biology

    08:15
    Plasmid Purification

    Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study.

    In this video, a step-by-step generalized procedure is described for how to perform plasmid purification. Plasmid purification includes three basic steps: growth of the bacterial culture, harvesting and lysis of the bacteria, and purification of the plasmid DNA. The video contains an explanation where the plasmid can be found in each step of the protocol and to quantitatively and qualitatively analyze plasmid DNA with a spectrophotometer and/or gel electrophoresis. There are different types of plasmid purification methods available, which are geared toward desired yield, plasmid copy number, and bacterial culture volume.

  • Basic Methods in Cellular and Molecular Biology

    10:05
    The ELISA Method

    An enzyme-linked immunosorbent assay (ELISA) is typically performed to detect the presence and/or amount of a target protein of interest within an experimental sample. Detection of the target protein is made possible by antibodies, which make the ELISA an immunoassay. Through a series of incubation and washing steps, these antibodies, which are frequently linked, or conjugated, to an enzyme, will detect protein coating the bottom of a well on a microtiter plate. When exposed to a substrate, antibody-bound enzyme will cause a color change, thereby indicating the presence of the protein-of-interest in the sample. In this video, the theory behind how ELISAs work is explained, including a discussion of both primary and secondary antibody binding and the importance of blocking steps. Theory is followed by practice, as the video progresses to an explanation of the step-by-step procedure. Finally, variations of the standard ELISA such as the sandwich and competitive ELISAs are introduced, and real world applications of this method, such as in over-the-counter pregnancy tests are explained.

  • Basic Methods in Cellular and Molecular Biology

    12:18
    Bacterial Transformation: Electroporation

    The term “transformation” refers cellular ingestion of foreign DNA. In nature, transformation can occur in certain types of bacteria. In molecular biology, however, transformation is artificially induced through the creation of pores in the bacterial cell walls. Bacterial cells that are able to take up DNA from the environment are called competent cells. Electrocompetent cells can be produced in the laboratory and transformation of these cells can be achieve via the application of an electrical field that creates pores in the cell wall through which DNA can pass. The video explains the equipment used in electroporation such as an electroporator and electroporation cuvette. The video also goes through a step-by-step procedure about how to create electrocompetent cells and electroporate cells of interest. Prediction of the success of a transformation of an experiment, by observing the time constant, as well as the importance of removing salt from the solutions when electroporating, are also mentioned.

  • Basic Methods in Cellular and Molecular Biology

    11:00
    Bacterial Transformation: The Heat Shock Method

    Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of bacteria. In molecular biology, transformation is artificially reproduced in the lab via the creation of pores in bacterial cell membranes. Bacterial cells that are able to take up DNA from the environment are called competent cells. In the laboratory, bacterial cells can be made competent and DNA subsequently introduced by a procedure called the heat shock method. Heat shock transformation uses a calcium rich environment provided by calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane. A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell. This video goes through a step-by-step procedure on how to create chemically competent bacteria, perform heat shock transformation, plate the transformed bacteria, and calculate transformation efficiency.

  • Basic Methods in Cellular and Molecular Biology

    07:28
    Separating Protein with SDS-PAGE

    Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their size and nothing else. SDS, an anionic detergent, is used to produce an even charge across the length of proteins that have been linearized. By first loading them into a gel made of polyacrylamide and then applying an electric field to the gel, SDS-coated proteins are then separated. The electric field acts as the driving force, drawing the SDS coated proteins towards the anode with larger proteins moving more slowly than small proteins. In order to identify proteins by size, protein standards of a known size are loaded along with samples and run under the same conditions. This video presents an introduction to SDS-PAGE by first explaining the theory behind it and later demonstrating its step-by-step procedure. Various experimental parameters, such as the polyacrylamide concentration and voltage applied to the gel are discussed. Downstream staining methods like Coomassie and silver stains are introduced, and variations of the method, like 2D gel electrophoresis are presented.

  • Basic Methods in Cellular and Molecular Biology

    09:21
    DNA Gel Electrophoresis

    DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of agarose, and within the gel, charge particles will migrate and separate based on size. The negatively charged phosphates of the DNA backbone cause DNA fragments to move toward the anode - a positively charged electrode.

    The video explains the mechanism by which DNA fragments are resolved on an agarose gel, and it provides a step-by-step generalized procedure for how to prepare agarose gels, load DNA samples, run a DNA gel, visualize DNA fragments, and properly dispose of the gel and running buffer after the experiment is concluded.

  • Basic Methods in Cellular and Molecular Biology

    13:27
    PCR: The Polymerase Chain Reaction

    The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling – cyles of temperature changes at fixed time intervals. Using a thermostable DNA polymerase, PCR can create numerous copies of DNA from DNA building blocks called dinucleoside triphosphates or dNTPs. There are three steps in PCR: denaturation, annealing, and elongation. Denaturation is the first step in the cycle and causes the DNA to melt by disrupting hydrogen bonds between the bases resulting in single-stranded DNA. Annealing lowers the temperature enough to allow the binding of oligonucleotide primers to the DNA template. During the elongation step DNA polymerase will synthesize new double-stranded DNA. This video provides an introduction to the PCR procedure. The basic principles of PCR are described as well as a step-by-step procedure for setting up a generalized PCR reaction. The video shows the necessary components for a PCR reaction, includes instruction for primer design, and provides helpful hints for ensuring successful PCR reactions.

  • Basic Methods in Cellular and Molecular Biology

    10:01
    Passaging Cells

    Cell lines are frequently used in biomedical experiments, as they allow rapid culture and expansion of cell types for experimental analysis. Cell lines are cultured under similar conditions when compared to freshly-isolated, or primary, cells, but with some basic important differences: (i) cell lines require their own specific growth factor cocktails and (ii) their growth must be more closely monitored than primary cells, as the mutations that allow them to be grown indefinitely also can quickly lead to their overgrowth. Therefore, when a cell line reaches the point of growth in culture where it covers most of the bottom of the culture container, or about a 90% confluency, the cells must be resuspended, washed, used experimentally, frozen for later use, or re-seeded for further expansion in new culture containers. This video will demonstrate how to use media indicators to determine cell culture health, which reagents and equipment are useful for safely removing adherent cell lines from culture, and various methods for transferring these robustly expanding cells into new cultures will be discussed. Also demonstrated are methods for how to culture feeder cells (important for providing essential growth factors to cell lines) and how to expand large numbers of cell line cultures at once.

  • Basic Methods in Cellular and Molecular Biology

    09:54
    Molecular Cloning

    Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant DNA fragments in host organisms. The DNA fragment, which may be a gene, can be isolated from a prokaryotic or eukaryotic specimen. Following isolation of the fragment of interest, or insert, both the vector and insert must be cut with restriction enzymes and purified. The purified pieces are joined together though a technique called ligation. The enzyme that catalyzes the ligation reaction is known as ligase. This video explains the major methods that are combined, in tandem, to comprise the overall molecular cloning procedure. Critical aspects of molecular cloning are discussed, such as the need for molecular cloning strategy and how to keep track of transformed bacterial colonies. Verification steps, such as checking purified plasmid for the presence of insert with restrictions digests and sequencing are also mentioned.

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