JoVE Science Education > Basic Methods in Cellular and Molecular Biology
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.
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. 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.
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.
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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 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.
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. There are different types of plasmid purification methods available, which are geared toward desired yield, plasmid copy number, and bacterial culture volume.
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.
Prokaryotic genomes: data retrieval
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. 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.
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. 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. All work should be done behind Perspex screens 1 cm thick to protect the operator from radiation. Usual precautions for handling 32 P-labelled chemicals must be observed at all times. Prepare nick translation mix in a 1.
All column equipment should be sterile. Equilibrate the column with column buffer. Monitor effluent with a Geiger Counter. The first peak is the labelled DNA. The second peak is 32 P-dCTP. The first peak is collected in a volume of ml. Single-stranded DNA immobilized on the nitrocellulose filter is hybridized to the single stranded radiolabeled DNA probe. Several methods are available for this reaction but, commonly, formamide or dextran sulphate are used.
Dextran sulphate, although viscous to handle, provides a reliable and sensitive assay method and is the method of choice described here. The procedure described here provides high stringency and only closely homologous DNA sequences will hybridize. If the DNA probe is unlikely to be closely related to the DNA under examination, the stringency can be lowered by either lowering the temperature or increasing the salt concentration i.
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Plastic bags and a plastic-bag sealer any commercially available system sold for domestic use is suitable. Place the nitrocellulose blot inside a plastic bag. Add solution A. Expel air bubbles. Seal bag. Cut off the corner of the plastic bag. Pour out solution A. Add solution B. Pour out solution B. Add solution C.
This is identical to solution C but contains in addition million cpm of 32 P-labelled DNA probe. Pour out solution C.
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Add solution D. After hybridization the Southern blot is washed to remove nonspecific hybridization. The important step in this procedure is step 3, which determines the stringency required. The conditions described here provide a high stringency wash.
If the DNA probe is not closely related to the DNA under examination, the stringency of the wash in step 3 should be lowered by either decreasing the temperature or increasing the salt concentration i. Remove the nitrocellulose filter from the plastic bag in which hybridization has taken place and put the filter into a plastic tray. Wash the blot with rinses at room temperature 1 minute each with ml each of solution made as indicated:. While the blot is still wet it should be scanned with a Geiger counter.
If a large amount of radioactivity is detected scattered all over the filter, there may be nonspecifically hybridized DNA still bound and the blot may need to be washed for longer repeat steps 3 and 4.