Preparation of electrocompetent cells and electroporation of e. In 1984, gibco brl now part of thermo fisher scientific became the first company to offer competent cells commercially, with the. We have examined bacterial electroporation with a specific interest in the transformation of large dna, i. Electroporation, originally developed as a method to introduce dna into eukaryotic cells 1, has subsequently been extensively used for bacterial transformation 2,3. For most bacterial species, the highest transformation efficiencies are obtained when cells are harvested in early to midlog growth. When dna is ad ded t o comp ete nt ce lls, some of the cells will take up the dna and are transformed. This protocol is for the typical electro transformation of e. This procedure is an effective method for the transfer of dna to a wide range of gramnegative bacteria, such as escherichia coli, and reports indicate that 10 9 electrotransformants per microgram of dna can be. Place electroporation cuvettes 1 mm and microcentrifuge tubes on ice. Autoclave two 500 ml centrifuge bottles for spinning down cells tomorrow. Since the development of artificial transformation of e.
Transforming plasmid dna into electrocompetent cells 1. Inoculate a single colony of corynebacterium or rhodococcus or similar strain into 25 ml of rich medium e. Electroporation, originally developed as a method to introduce dna into eukaryotic cells, has subsequently been extensively used for bacterial transformation 2, 3. Is there a commercial e coli strain that is better at cotransformation. What is the e coli transformation efficiency for 2 plasmids. 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. Effect of electroporation versus hanahan protocols on the transformation of escherichia coli hb101 with chromosomal dna from escherichia coli hb101, escherichia coli b23, and bacillus subtilis wb746 and the plasmid p328. Comparison of the transformation efficiencies achieved. Evaluation of parameters for high efficiency transformation.
Transformation of ecoli via electroporation request pdf. Cells that can readily take up dna are referred to as competent cells. Section 3 electrical variables the electrical conditions for the electroporation of e. Using electroporation to transform escherichia coli results in transformation efficiencies greater than can be obtained using the best chemical methods. Bacterial transformation and competent cellsa brief. By subjecting mixtures of cells and dna to exponentially decaying fields of very high initial amplitude, we routinely obtain 10 9 to 1010 trans. An investigation into the relative efficiency of e. The nature of the transformation process in escherichia. Handling bacteria also requires that one practice aseptic technique.
Cells are placed in suspension in an appropriate electroporation buffer and put into an electroporation cuvette. For control electroporation dilute puc19 to 10 pgl with milliq water. Electrocompetent cells transformation protocol neb. If want to cut at xbai or other dam enzyme site, use scs110 cells. A highvoltage current is applied to the cells, which temporarily permeabilizes the plasma membrane and allows dna or. Recently, electroporation, or electropermeabilization, in which a brief high voltage electric discharge is used to render cells permeable to dna, has revolutionized the transformation of bacteria. Transformation by electroporation electroporation theory electroporation or electropermeabilization a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by externally applied electrical field. C plasmid dna recombinant plasmid pure h 2 o sob agar plates containing 20mm mgso 4, and the appropriate antibiotic soc medium method. Chemical transformation is more convenient and electroporation is more efficient.
A highvoltage current is applied to the cells, which temporarily permeabilizes the plasma membrane and allows dna or other small molecules to enter. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from genlantis. Studies on transformation of escherichia coli with plasmids. Transformation efficiency should be determined under conditions of cell excess. It is easy to obtain transformation efficiencies 10 8 per milligram dna and efficiencies of 10 10 have been reported. For incubation on ice, make sure the tubes are standing in an icewater mix, because without water, the cooling effect of ice is not reproducible due to the air between the ice fragments, especially if you have to incubate for a certain period of time. In cloning protocols, artificial transformation is used to introduce recombinant dna into host bacteria e. Download a pdf containing pricing for our full product list. Chassy, annick mercenier and jeannette flickinger the introduction of dna into bacteria by transformation is an essential step in the construction of recombinant strains.
Step 1 prepare competent cells as per the competent cells for electroporation protocol step 2 aliquot a small amount, around 1. Each 2 ml on culture will give you 200 l of cells, or 4 electroporations. This protocol is for the typical electrotransformation of e. 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. The introduction of dna into bacteria by transformation is an essential step in the construction of recombinant strains.
Protein extraction by means of electroporation from e. Use one cuvette for each dna sample you are transforming. The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. Get a printable copy pdf file of the complete article 1. Artificial transformation encompasses a wide array of methods for inducing uptake of exogenous dna. Neb turbo, neb 5alpha and neb 10beta competent li strains are available as electrocompetent cells. For incubation on ice, make sure the tubes are standing in an icewater mix, because without water, the cooling effect of ice is not reproducible due to the air between the ice fragments, especially if you have to incubate for a certain period. It is preferred over the chemical method for transferring exogenous dna into. Transformation is a key process in molecular cloning, by which multiple copies of recombinant dna molecules are produced. Plasmid dna transformation in escherichia coli 563 containing media plates.
Library diversity can be limited by the transformation efficiency of the e. We have demonstrated that conditions maybe selected which increase the average size of bac clones generated by electroporation and compare the overall. Keywords electroporation 4 escherichia coli 4 protein extraction 4 bacterial inactivation. Dialyze your dna samples using a nitrocellulose filter and di water. Electrotransformation efficiency is function of many factors which include 1 number of cell washes prior to electroporation, 2 electroporation cell number, 3 electroporation dna amount, and 4 cell growth phase. Heat denatured ligation reactions can be used for electroporation directly. Dh5 revised 22496 before starting procedure, preparechill the following. The main advantage of electroporation is its applicability for transient and stable transfection of all cell types. Acinetobacter baumannii is an emerging, nosocomial pathogen that is poorly characterized due to a paucity of genetic tools and methods. Grow cells with vigorous aeration overnight at 37c.
Transformation by electroporation conditions are for e. Bacterial transformation protocols find more protocols and selection guides in the molecular biology guide. Cacl 2 treatment of the recipient cells is absolutely necessary for transformation and the optimum concentration was found to be 30 mm. The nature of the transformation process in escherichia coli. Electroporation is an ionic restricted physical process that requires a cell suspension of high resistance and very low conductivity for a high degree of success dower et al.
Electroporation is already an established technique in several areas of. For the preparation of electrocompetent cells follow this protocol note. Transformation of escherichia coli with large dna molecules by. Once dna is added to the cells, electroporation can be carried out immediately. The most common type of competent bacteria that is transformed in molecular biology research is e. The consensus electroporation protocol should be consulted if deviating from the procedure outlined here. We have used dna from bacterial artificial chromosomes bacs ranging from 7 to 240 kb, as well as bac ligatlon mixes containing a range of different sized molecules. Used in molecular biology as a way of introducing some substance inside the cell. A library of peptides or proteins is expressed or displayed on the surface of bacteriophage particles viruses that infect bacteria, and screened for ability to bind to the target of interest. The efficiency of transformation is dependent upon temperature during incubation of.
Whenever handling bacteria, make sure the work area is as clean as possible. Eukaryotes include protozoa such as giardia which causes giardiasis, a severe diarrhea, fungi such as unicellular yeasts which cause oral thrush, and algae such as bluegreen algae. Updated 21811 transformation by electroporation conditions are for e. In this protocol cells are made competent by washing them in 10% glycerol. Remember to count for controls, and making extra is a good idea. Those factors have limitedly been concomitantly investigated in. Are any studies that have looked at the correlation between number of plasmids transformed and transformation efficiency. We have examined bacterial electroporation with a specific interest in the. Clean and dry electroporation cuvettes throroughly on the cuvette washer. Transforming plasmid dna into electrocompetent cells. This appendix describes a procedure for electroporation that can be used to transform many different types of bacteria.
Electroporation cuvettes and microcentrifuge tubes should be prechilled on ice. Competent cells can be stored at 80o c until they are needed. The following tips will help maximize transformation efficiencies when using electrocompetent cells from neb. Transformation protocol using heat shock mft, 112103 1 take competent li cells from 80oc freezer. Effect of electroporation versus hanahan protocols on the. Transformation of escherichia coli with large dna molecules. Prepare 17 mm x 100 mm roundbottom culture tubes e. This procedure prepares glycerol stock cultures of bacteria for electroporative transformation.
This procedure is an effective method for the transfer of dna to a wide range of gramnegative bacteria, such as escherichia coli, and reports indicate that 10 9 electrotransformants per microgram of dna. Transformation was performed by electroporation of 500 ng dna 10 l ligation into a 60 l suspension of electrocompetent e. Gyt medium ice cold glycerol 10% vvmolecular biology grade, ice cold lb medium, prewarmed to 37. Transformation of bacteria by electroporation sciencedirect. For chemical transformation, cells are grown to midlog phase, harvested and treated with divalent. These plates were incubated at 37c overnight and the resulting transformant colonies were scored and analyzed. Volker briese, universitatsfrauenklinik rostock electroporation can be used for both transient and stable transfection of mammalian cells. While whole genome sequence data from several epidemic and.
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