Transfection generally refers to the introduction of foreign dna into bacterial andor mammalian cells. Through this way, pores appear in the surface of the living structure and biological material can pass through it easily. Add 3 mg of binary vector into the agrobacterium cells. Electroporation using squarewave generating devices, like lonzas nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this. This protocol is for the typical electrotransformation of e. Electroporation is one of the most widespread techniques used in modem molecular genetics.
If bluewhite screening for recombinants is required, incorporate 1 mm iptg, 300 gml sgal or 40 gml xgal and 500 gml ferric ammonium citrate in the lb agar. Each 2 ml on culture will give you 200 l of cells, or 4 electroporations. Clean and dry electroporation cuvettes throroughly on the cuvette washer. Because of the small cell size, high eld strengths are required i. Abstract in this chapter, basics and mechanisms of electroporation are presented. Introducing binary plasmid into agrobacterium by electroporation. Electroporation cell fusion instruments biocompare. Read the instruction manual before using the gene pulser xcell electroporation system. One important variable is the choice of electroporation bufferthe appropriate buffer can increase cell transfection efficiency 5098%.
The principle application of electroporation is focused on vertebrate tissue and organisms. Depending on the antibiotic marker present in the plasmid dna, incorporate appropriate antibiotic in the lb agar. Current protocols in microbiology wiley online library. Hydrolytic activity of bacterial supernatants for fungal suppression as the fungal growth suppression by biocontrol agents bca in solid media using dual plate assay has some issues regarding nutrient limitation. Electroporation protocols microorganism, mammalian. Electroporation protocols for microorganismsedited by jac a. Researchers can also use chemical or manual methods of aligning the cells. Universal electroporation buffer is provided to increase cell transfection efficiency. Development of an electrotransformation protocol for genetic. The system consists of a pulse generator module, a shocking chamber, and a cuvette with incorporated electrodes figure 1. Blog design your own electroporation protocol episode 8. The neon transfection system offers an innovative electroporation transfection method that utilizes a proprietary biologically compatible pipette tip chamber to generate a more uniform electric field for a significant increase in transfection efficiency and cell viability. Transforming plasmid dna into electrocompetent cells.
Electroporation protocols for microorganisms jac a. Recently, electroporation, or electropermeabilization, in. Among the many described possibilities, silica nanoparticles sinps represent a good choice because of their ease of synthesis, the. To date, very few applications of electroporation in highcontent and phenotypic screening have been published. It is most commonly used to introduce dna into cells for investigations of gene structure and function, and in this regard, electroporation is both highly versatile, being effective with nearly all species and cell types, and highly efficient.
Even as many bacteria, mammalian, plant, yeast and insect cells have been successfully electroporated, researchers are still improving the process. Remember to count for controls, and making extra is a good idea. Ensure there are a clean pipette bulb and sterile, prechilled long pasteur pipettes to hand. Pdf an efficient electroporation protocol for the genetic. The efficiency of transfection by electroporation is dependent upon cell type. Protocols employing millisecond pulses are more effi cient than. Using a passive, selfregulating 4step mechanism, discrete volumes of two fluids are alternatingly issued into a common intermediate chamber. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. Current protocols in microbiology is a comprehensive source of practical methods for designing and implementing successful experiments in microbial research across viruses as well as unicellular and cellcluster organisms including prokaryotes bacteria, archaea and eukaryotes fungi, protists, microalgae to study their genetic, biochemical, and biophysical properties. Generating mutants in these recalcitrant bacteria, while not.
Storing and handling recombinogenic bacteria strains. Using electroporation to transform bacteria, yeast and. During the 1980s, several studies on the use of electroporation for. The successful electroporation procedure is predicated by the cell properties, cell environment and the applied electric fields.
This protocol describes a method for transforming agrobacterium with plasmid dna using electroporation in a manner similar to that commonly used for escherichia coli. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed. Design your own electroporation protocol episode 8. Make a 16 fold dilution of the on culture growth medium gm, incubate in 37 degc shaken at 200rpm.
For fibroblasts, which are easily transfected by calcium phosphate or deaedextran coprecipitation units 9. Electroporation is one of the most widespread techniques used in modem. Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Materials free fulltext increasing uptake of silica. Authoritative and easily accessible, electroporation protocols. Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or dna to be introduced into the cell also called electrotransfer. A novel electroporation system for efficient molecular delivery into chlamydomonas reinhardtii with a 3dimensional microelectrode skip to main content thank you for visiting. Kadlec et al application of pulsed electric field on bacteria the high voltage sourcecan provide quasirectangular shape of pulse up to 60 kv of amplitude, 2 s fwhm and 50 hz repetition rate. Pdf in this chapter, basics and mechanisms of electroporation are presented.
In recent years electroporation has become the preferred method since it is very efficient and relatively easy to perform. Transformation of agrobacterium using electroporation. However, some cell lines remain difficult to transfect, and. Supplement 14 current protocols in microbiology f or 7h9 powder, the appr oximate amounts of the components per 900 ml are.
Combine the cell pellets and wash the cells in one bottle as in step 5. Microorganism, mammalian system, and nanodevice, third edition aims to be an invaluable resource. Cell sizespecific transfection by micropillar array electroporation. To improve transfection, several methods have been developed.
Development of an electrotransformation protocol for. The electroporation protocol for each cell line is summarized in table 1. Full text get a printable copy pdf file of the complete article 1. Transfection is an important tool used in studies investigating gene function and the modulation of gene expression, thus contributing to the advancement of basic cellular research, drug discovery, and target validation. Microorganism, mammalian system, and nanodevice, third edition aims to be an invaluable resource for investigators both in and outside of this field. Miller introduction electroporation is a versatile method for the introduction of dna into bacteria.
To correct for cell number after a variety of metabolic assays i perform in my organoids, i am looking for a quick and easy cell number assay that can be performed in a 96 well plate. General protocol for optimization of electroporation. This outcome is in agreement with other clostridial electrotransformation protocols that employed phosphatebuffered sucrose as the wash and electroporation buffer, as all such protocols utilize isotonic rather than hypertonic sucrose 25,26,4446,58,59. A novel electroporation system for efficient molecular. Also it is limited to bacterial, yeast and plant protoplasts while electroporation can be applied to mammalian cells. Compare electroporation cell fusion instruments from leading suppliers on biocompare.
Electroporation protocols for microorganisms springerlink. For some wild type strains such as ybt1520 and ctc, using such protocols resulted in electroporation. Transformation of bacteria by electroporation bruce m. Remove from 80 o c tubes containing 80 ml of electrocompetent agrobacterium tumefaciens strain lba4404, or gv3100 cells. The consensus electroporation protocol should be consulted if deviating from the procedure outlined here. An efficient electroporation protocol for the genetic. The first seven chapters of electroporation protocols for micro organisms. Furthermore, because electroporation is easy and rapid, it is able to transfect a large number of cells in a short time once optimum electroporation conditions are determined.
Dialyze your dna samples using a nitrocellulose filter and di water. Electroporation protocols for agrobacterium springerlink. We use a biorad micropulser with the conditions noted above. Electroporation protocols for microorganisms ebook, 1995. Preparation of electrocompetent bacteria electroporation of. I am following some electroporation protocols found in the literature. Protocols employing millisecond pulses are more effi. When the field strength of the electroporation pulse is high enough, reversible permeability occurs in the cell membrane that allows outside molecules to enter the cell. Electroporation works well with cell lines that are refractive to other techniques, such as calcium. Generally, buffers with low ionic constants enhance transfection efficiency. Transfection protocol library the transfection protocol online library contains protocols obtained from the literature, developed by biorad scientists, or submitted by scientists like you.
For more information about electroporation protocols, see. The efficiency of irreversible electroporation can be monitored by plating the treated sample on nutrient agar and counting the number of colonyforming units cfu. Electroporation method use an electrical field to create transient pores in cellular membrane which enables the intracellular delivery of charged molecules for example oligonucleotides, dna, rna, sirna, proteins to the cytoplasm and nucleus of the targeted cells. Set up the electroporation conditions prior to placing the cuvette into the electroporation chamber. Electroporation is the application of controlled electrical pulses. It is not exhaustive and as always there will be many variants of the methods used by others. Volker briese, universitatsfrauenklinik rostock electroporation can be used for both transient and stable transfection of mammalian cells. Transformation of bacteria by electroporation sciencedirect. Electroporation of microorganisms electroporation is widely used to transform bacteria, yeasts and other microorganisms with plasmid dna. Transforming plasmid dna into electrocompetent cells 1. Electroporation is the process of biotechnology to pass the electric current through the living surface fro example, a cell or a molecule. Place electroporation cuvettes 1 mm and microcentrifuge tubes on ice. Electroporation is a commonly employed method for introducing dna into bacteria in a process known as transformation. General protocol for transformation of bacteria 3 3.
Chassy, annick mercenier and jeannette flickinger the introduction of dna into bacteria by transformation is an essential step in the construction of recombinant strains. Welcome to the eighth post in our series providing tips for developing and improving your own electroporation methodguidelines for mammalian cells. The protein extraction by means of electroporation ef. The main properties of the cells which affect the electroporation are cell size, shape, membrane structure, and surface charge 2, 12.
The main advantage of electroporation is its applicability for transient and stable transfection of all cell types. This is the protocol for making two 250 ml cultures of electrocompetent cells. Nonviral methods like liposomes and electroporation. Preclinical and clinical gene medicine, second edition provides indepth knowledge on the delivery of naked dna and smallinterfering rna sirna to the targeted cells, tissues, and animals for prevention and treatment of disease. Recombineeringbased procedure for creating bac transgene. Experimental protocols this file is a collection of experimental protocols for molecular microbiology and molecular plantmicrobe interactions. These pulses are delivered to a pair of electrodes by a pulse generator. Electroporation efficiency in mammalian cells is increased. Electroporation is firmly established in the armamentarium of transfection techniques that include viral vectors, chemical or reagentbased methods, and mechanical gene delivery. Efficiency up to 90% transfection and geneediting efficiency in.
Electroporation electrofusion products harvard apparatus. With chargepulse switch of the electroporator in the pulse position, connect the leads from the power supply to the corresponding coloured terminals on the back of the electroporator. To our knowledge, this study presents the first demonstration of dna transfer into c. Unraveling the unseen players in the ocean a field guide to water chemistry and marine microbiology andreas florian haas 1, ben knowles 1, yan wei lim 1, tracey mcdole somera 2, linda wegley kelly 1, mark hatay 1, forest rohwer 1. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. The first seven chapters of electroporation protocols for micro organisms describe the underlying theory of electroporation, the com mercially available instrumentation, and a number of specialized electroporation applications, such as cdna library construction and interspecies dna electrotransfer. Electroporation protocols for microorganisms book, 1995. In general, efficient transfection is achieved at higher. Search for library items search for lists search for contacts search for a library. Autoclave 1 l sob and 3 l ddh2o, and autoclave or filter sterilize 10 ml 10% glycerol. The study of microorganisms such as fungi, bacteria, algae, archaea, and viruses. The high voltage source was designed to work to matched load of approximately 2 k.
Although the transformation efficiency for agrobacterium is lower than that for e. Pulsed electrical fields can be used to introduce dna into a wide variety of animal cells 1,2. Electroporation of cells in complex materials and tissue l. Electroporation protocols, buffers, kits electroporation. Please use one of the following formats to cite this article in your essay, paper or report. On the other hand, heat shock leads to lower transformation efficiencies than electroporation and takes longer. In the fields of biology and medicine, nanoproducts such as nanoparticles nps are specifically interesting as theranostic tools, since they offer the double capacity to locally deliver active drugs and to image exactly where the product is delivered. It is most commonly used to introduce dna into cells for investigations of gene structure and function, and. Electroporation is a powerful transfection tool useful for studying gene function. Although a number of electroporation devices are available for the use in 96well and 384well plate format, work has been focused on establishing and optimizing protocols for the electroporation of otherwise difficulttotransfect cells. Frontiers an efficient electroporation protocol for the. Electroporation protocols, buffers, kits applications.
Electroporation of a plasmid into dh10b and its derivatives. Protein extraction by means of electroporation from e. Elaboration of an electroporation protocol for large. In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by. For optimal results, proper mixing is essential and should be done as follows. Prepare an on culture of bacillus subtilis 168 lb media with appropriate antibiotic incubate at 37degc.
Biolabprotocols includes animal techniques, biotechnology, biochemistry, cell biology, microbiology, molecular biology, immunology, plant biology, general lab techniques, troubleshooting tips, research methods and all other laboratory protocols. Chassy, annick mercenier and jeannette flickinger the introduction of dna into bacteria by transformation is an essential ste p in the construction of recombinant strains. One nanogram of uncut supercoiled vector dna is recommended to be transformed in parallel with insertpgex ligations to determine the efficiency of each competent cell preparation. Transfection of mammalian cells by electroporation.
Methods in molecular biology 47 electroporation protocols for. In vivo electroporation, which uses living animals, is quickly performed with relatively simple equipment, compared with generation of transgenic and gene knockin mice and construction of recombinant viruses, which require longer time and special. Transferring useful genes into bacteria in order to transform them into the target plant is an essential part of the process, which can be achieved by electroporation. Genetic transformation and complementation ncbi nih. Delivery of genetic material in tissues using electric pulses for treating diseases is known as electroporation gene therapy. The pc module is recommended for the electroporation of bacteria and fungi using exponential. Dilute a 10 ml overnight lb or 2xyt culture into 1 l sob without mg or glucose and growing until od550 of 0. Get microbial systems biology methods and protocols pdf file for free from our online library pdf file. The micropulser system is used for the electroporation of bacteria, yeast, and other microorganisms where a high voltage electrical pulse is applied to a sample suspended in a small volume of high resistance media. It is most commonly used to introduce dna into cells for investigations of gene structure and function, and in this regard, electroporation is both highly versatile, being effective with nearly all. In addition to the electroporation protocol, we also identified effective antibiotic selection systems and origins of replication capable of sustaining plasmids in c. There are preset protocols for bacteria, fungi, and mam.
Efficient procedure and methods to determine critical. Using genepulser x cell from biorad and cuvettes 0. The following texts are the property of their respective authors and we thank them for giving us the opportunity to share for free to students, teachers and users of the web their texts will used only for illustrative educational and scientific purposes only. We will refer to protocols that by themselves, without electrolysis, do not cause cell death, as reversible electroporation protocols. During that period many of the bacteria that cause human disease were identified and characterized. Electroporation is an efficient method to transfect dna and rna into a variety of cell types. Prepare 17 mm x 100 mm roundbottom culture tubes e. Therefore, bacterial transfection protocols focused little on cell viability after electroporation and this is likely to have caused electroporation to be conceived as a rather violent means of achieving gene transfer.
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