Work sterile. Agrobacterium Transformation and Competent Cell Preparation Monday, January 07, 2013 3:59 PM Methods Page 1 . What is the significance of recognition sequences of restriction enzymes? What actually happens when cells are "competent"? This step is repeated at least once more. Such cells are said to be "competent." i think it is helpful . To make it clear what I'm talking about, I use a protocol like the following: Take cells out of -80C and thaw on ice for 5 min. The bacterial cells were treated with calcium chloride... Electroporation: In this technique, cells are subjected to an electric field to increase their permeability. Most of the cells cannot take up unless they have been exposed to certain physical or chemical treatments. What governs transformation efficiency (besides obvious things like amount of DNA or cells)? To avoid this verification in future, please. PreserveArticles.com: Preserving Your Articles for Eternity. In the evening pick a single colony and transfer into 5 ml LB broth. Calcium chloride transformation technique is the most efficient technique among the competent cell preparation protocols. What is the role of CaCl2 in the preparation of competent cells? What does the calcium chloride do? Treatment with calcium ions is the standard method for the preparation of those cells. Harvested cells are then processed according to the method of transformation, whether by heat shock or electroporation (Figure 2). Suitable antibiotic (e.g. (iii) Transfer the culture into sterile centrifuge tubes in a laminar air flow. What is the role of CaCl2 in the preparation of... Email me at this address if a comment is added after mine: Email me if a comment is added after mine. Precool Centrifuge with 500 ml bottle adaptors to 4°C 8. Autoclave: … Our mission is to liberate knowledge. Rubidium Chloride Competent Cell Protocol.pdf: 33.02 KB: Protocol. (vi) Add 50 µl, 100 (il and 200 µl of transformed E. coli cells separately [obtained after step (iv)] to three different plates. Content Guidelines Grow O/N @ 37°C. (i) Add 5 µl of the plasmid DNA (about 10-50 nanograms) to 100 pi of competent cells prepared. Learn more about transformation and how it is used in cloning workflows. Consequently, efficiency of receiving the foreign DNA is increased. Complete information on Photovoltaic Cells (Solar Cells), Get Complete Information on DNA Sequencing, Controlling in Management # Meaning, Definition, Types, Process, Steps and Techniques. By gently raising the temperature to 42°C, the uptake of foreign DNA by E. coli cells is stimulated. Structural Organisation in Animals and Plants, Application of Biotechnology in health and agriculture. Two widely used chemical methods involve treating bacteria with calcium chloride or hexamine cobalt and subjecting the cells to a heat shock. Similarly, transfer the non- transformed competent E. coli cells on antibiotic-containing LB medium as control to rule out the contamination. Essay on Plating and Culture of the Transformed Host Cells, Get Complete Information on Insulin and Cloning of Insulin Gene. Add 6 ml of ice-cold 0.1 M calcium chloride. The cells are incubated on ice with the DNA, and then briefly heat-shocked (e.g., at 42 °C for 30–120 seconds). The following preparation should be done in advance: Streak E. coli suspension onto the surface of fresh LB plate so that a single colony may be obtained. This is done by creating temporary holes in the cell membrane by various methods. What is the role of nucleolus in the cells actively involved in protein synthesis? This allows the transformation to occur. Incubate the plate at 37°C overnight. Keep the eppendorf tube in the water bath in such a way that the competent cells should be immersed for 2 minutes. Hence, in order to introduce foreign DNA efficiently into these cells, the cell should have to undergo a chemical treatment. The control plates show no colonies on which competent cells containing no plasmid DNA were spread. Streak out frozen glycerol stock of bacterial cells (Top10, DH5α, etc.) PreserveArticles.com is an online article publishing site that helps you to submit your knowledge so that it may be preserved for eternity. Thaw the competent cells on ice if they are stored frozen. • 9. Tips, micropipettes, centrifuge tubes. (vii) Incubate the plates at 37°C overnight and observe them on the next day. Together, these ease the passage of DNA through the hydrophobic cell membrane. Disclaimer Transformed cells will allow for downstream applications such as plasmid … Thus, the DNA can then pass through the cell on subsequent heat shock treatment. • 8. By addition of CaCl2 solution, Ca2+ ions destabilize the cell membrane or also form a complex with the foreign DNA which attaches to the cell surface. E. coli Calcium Chloride competent cell protocol 1. It increases the ability of a prokaryotic cell to incorporate plasmid DNA allowing them to be genetically transformed. ... Calcium Chloride 75 mM 147.02 Glycerol 15%. Grow plate overnight at 37°C. Cells stored at -80 o C can be used for transformation for up to ~6 months NOTE: through the process, cells should be treated with care. Resuspend the cells with 2.5 ml of ice-cold 50-mM CaCl2. Hence, E.coli cells can be made more competent in laboratory by treating an ice-cold solution or CaCl 2, rubidium chloride or the other salts. Heat-shocking facilitates the transport of plasmid into the competent cell. When cells are ready to harvest chill flasks on ice for 15 - 30 minutes PreserveArticles.com is a free service that lets you to preserve your original articles for eternity. The other plates show colonies on which competent cells transformed with the plasmid were spread. Making Calcium Competent Cells Day 1 1. 2. (ii) Then quickly keep the culture flask on ice in a refrigerator for 10-20 minutes. (vii) In a laminar air flow discard the supernatant and re-suspend the cell pellets gently in 0.5 ml of ice-cold 0.1M CaCl2. Optionally, if required to store the competent cells for a longer period, resuspend the cells with 2.5 ml ice-cold 50-mM CaCl2 containing 10 % glycerol. DNA into the host cell and it is the topic of the discussion of today’s lecture. During 1970, it was found that E.coli cells soaked in ice cold solution were more efficient in receiving foreign DNA than the treated normal cells. antibiotic resistance markers. Cells are made competent by a process that uses calcium chloride and heat shock. 5. Copyright. Mix well and pour into Petri plates. LabBench Activity Competent Cells. Gently tap and keep the eppendorf tube on ice for 20 minutes. 4. Shake @ 37°C for 1.5-3hrs. Such chemically treated cells are called competent cells. Privacy: Your email address will only be used for sending these notifications. The competent cells can be prepared artificially in two ways, namely: Calcium Chloride: This method was proposed by Higa and Mandel. All the articles you read in this site are contributed by users like you, with a single vision to liberate knowledge. Under normal conditions several bacteria like E. coli receives a limited amount of DNA. Email me at this address if my answer is selected or commented on: Email me if my answer is selected or commented on. Transformation of Competent E. Coli Cells: (All steps should be carried out inside the laminar airflow). This is because the Ca ions being positively charged attack both the negatively charged DNA … Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning. Protocol used for the Lab Job of making competent cells. Grow cells to an OD 600 nm of 0.5 - 1 7. Develop the competent E. coli cells as described below: A. Decant the medium from the cell pellets. Then, incubate cells on ice for 30 minutes. Kanamycin‐resistant colonies appeared after approximately 7–14 days of incubation, as shown in Figure 2a Chemically competent cells are calcium chloride treated to facilitate attachment of the plasmid DNA to the competent cell membrane. Grow the culture to get the 0.3-0.5 OD at 600 nm (A600) (it takes 2-3 hours). The plasmid solution should be less than 5 microliters. Keeping the tubes on ice bucket, suspend the cell pellet gently in CaCl2. Background Information: Natural ability of a cell (either bacterium/yeast or mammalian cell) to take up cell free DNA present in extracellular environment is low Hence, E.coli cells can be made more competent in laboratory by treating an ice-cold solution or CaCl2, rubidium chloride or the other salts. Transformation is the process by which bacteria are made to take up exogenous DNA. Adjust pH … Under these conditions, the Ca2+ ion is thought to create pores in the membrane, assist binding of the DNA to the cell membrane and mask the negative charge on the DNA. Preparation of E.coli competent cells and transformation of these cells with a given plasmid. The addition of calcium chloride to a cell suspension promotes the binding of plasmid DNA to lipopolysaccharides (LPS). (viii)Transfer 100 pi of the above competent cells into 5 eppendorf tube (care should be taken for all transfer work to carry out on ice and in the laminar air flow). Transfer the bacterial cells to sterile, disposable, ice-cold 50ml polypropylene tubes. In calcium chloride transformation, the cells are prepared by chilling cells in the presence of Ca 2+ (in CaCl 2 solution), making the cell become permeable to plasmid DNA. The treatment using Calcium chloride (CaCl 2) is one such method of preparation of competent cells. Day 2 1. (v) Prepare LB plates containing streptomycin or ampicillin or any other suitable antibiotic depending upon the plasmid used (melt the autoclaved LB-agar medium and allow it to cool. It increases the bacterial cell’s ability to incorporate plasmid DNA, facilitating genetic transformation. streptomycin), tryptone, eppendorf tubes. When desired, the cells are thawed and DNA is added. The transformed E. coli cells can be placed on an LB plate containing appropriate antibiotic (depending upon the antibiotic resistance gene present on the plasmid DNA used for transformation). It was observed that a period of 24 h incubation in cold calcium chloride makes the bacterial cells 20–30 times more competent and 4–6 times more efficient for transformation as compared to the cells that are obtained immediately after CaCl 2 treatment (Blattner et al., 1977; Dagert and Ehrlich, 1979). 3. (vi) Centrifuge cells at 6,000 rpm for 8 minutes at 4°C, if possible. Positively charged calcium ions (Ca2+) attract both the negatively charged DNA backbone (phosphate) and the negatively charged groups in the lipopolysaccharides inner core. 2. Recover the cells by centrifugation at 2700g at 4°C for 10 minutes . In this method calcium chloride is used and can be performed in less than 3 hours. (y) Discard the supernatant in a laminar air flow. iv. 2. During incubation in water-bath temperature should be maintained accurately. So it is necessary to brought cells into log phase before the procedure is begun. what is role of glycerol is used in preparation of competent cells ? (ii) Maintain the temperature of a water-bath at 42°C. Transformation. Overview of competence and heat shock Rapidly growing cells are made competent more easily than cells in other Growth stages. Role of mgcl2 in competent cell preparation 2 See answers pihu1034 pihu1034 Explanation: the addition of calcium chloride to cell suspention promote the bidding onplasmid DNA lipopolysaccharides LPS. After the final wash, resuspend cells in a cold 50mL 0.1 molar calcium chloride plus 15% glycerol solution. i. E. coli host strain, Plasmid DNA, sodium chloride, yeast extract, ii. There are two main methods for the preparation of competent cells.They are Calcium chloride method and Electroporation. Incubate the resuspended cells on ice for 20 min. > high efficiency transformation – automation friendly competent cells Chemical transformation is achieved by suspending the cells in an ice-cold buffer that contains calcium chloride and other salts. What are the commonly used vectors for transformation in plant cells? Heat-shock transformation: Competent cells are chemically prepared by incubating the cells in calcium chloride (CaCl 2) to make the cell membrane more permeable [1,2]. Chemically competent cells were prepared using R. sulfidophilum cells by calcium chloride treatment. B. This method works very well for circular plasmid DNA. One is by suspending the bacterial cells in a high concentration of calcium chloride placed on ice. Some cells got to be exposed to some chemical or electrical treatments to transform them into competent cells. Typically, these cells are stored frozen. CaCl 2 is known to increase the efficiency of DNA uptake to produce transformed bacterial cells. Before publishing your Article on this site, please read the following pages: 1. Exponential phase cells are harvested & treated with cold calcium chloride, which renders the cell competent or suitable for taking up DNA . Naturally some competent cells have membrane proteins which collectively helps in uptake of DNA. Add to 100 microliters of the competent cells appropriate amount of plasmid solutions in TE. (iv) Pour 1ml of LB medium to the eppendorf tube and incubate the culture for 1 hour at 37°C. What is the origin of replication in DNA? The divalent Ca 2+ ions supposedly create transient pores on the bacterial cell wall by which the entry of foreign DNA is facilitated into the bacterial cells. This is because the Ca ions being positively charged attack both the negatively charged DNA and also the lipopolysaccharide membrane. Chemically competent cells were mixed with plasmid DNA and preincubated overnight without kanamycin followed by incubation with kanamycin. 1. 8:00am will be ready hopefully by 3:00pm 6. Development of Competent E. Coli Cells: (i) Pour 1 ml of the pure culture obtained on 3rd day as given in step (ii) into 100 ml LB medium and incubates at 37°C on a shaker (200-250 rpm). Inoculate a single colony into 25mL LB in a 250 mL bottle in the morning. Calcium chloride ( CaCl2) transformation is a laboratory technique in prokaryotic (bacterial) cell biology. Shake @ 37°C for 4-6 hrs. Principle of Competent Cells Competent cells have altered cell walls that allow the DNA to simply undergo it. (iv) Centrifuge at 6,000 rpm for 8 minutes at 4°C (a refrigerated centrifuge is preferred). What are the Common Methods of Gene Transfer into Host Cells? The competence of a bacterial cell for transformation could be artificially induced by exposing cells to calcium chloride prior to the addition of DNA. The treatment using Calcium chloride (CaCl2) is one such method of preparation of competent cells. the positive charge calcium ions attract negative charge DNA backbone and nagatively charge group in LPS inner core . Lab experiment 37.1: Preparation of chemically (CaCl. Then cells growing on such medium are selected and purified. Cycles of spinning and resuspending cells are often referred to as washing your cells. As a control, competent cells that have not been transformed with the plasmid DNA should also be plated onto a plate to rule out the contamination of cells. Agar, sodium hydroxide, calcium chloride, iii. The uptake of foreign DNA by Escherichia coli can be induced either through electroporation, which involves discharging an electrical voltage across bacterial cell membranes, or by making bacteria competent through chemical methods. Privacy Policy 3. Or 1. Cool the cultures to 0°C by storing them on ice for 10 minutes. E. coli cells are more likely to incorporate foreign DNA if their cell walls are altered so that DNA can pass through more easily. The competent cell is alternatively heated in a water bath, this opens the pores of the cell membrane allowing entry of the plasmid. Competent cells are the cells that can take up foreign DNA easily since they have altered cell walls. Keep the centrifuge tubes on ice for 30 minutes. The Hanahan or calcium chloride method is used to generate chemically competent cells. Thus, both the negatively charged DNA backbone and LPS come together and when heat shock is provided, plasmid DNA passes into the bacterial cell. (iii) After heat shock quickly remove the eppendorf tube and place it on ice for 2 minutes. No vortexing or excess pipetting should be performed, specially when the cells have been resuspended in CaCl 2 because lysis will result, decreasing the amount of competent cells). Then, a heat shock is given to the ice cold mixture which allows the DNA to enter the cells and then it is replaced on ice. onto an LB plate (no antibiotics since these cells do not have a plasmid in them). Collect the cell pellets by centrifugation at 6000 rpm for 5 min at 4 °C. This helps the bacteria to recover from the heat shock and show antibiotic resistance. Inoculate a single colony into 5mL Lb in 50mL falcon tube. 2. Incubate the tube at 37°C on a shaker (200-250 rpm) for about 16 to 18 hours. The cells resuspended in 100-150 microliters of the calcium solution are used for transformation. Prepare 2000 ml of 50 mM Calcium chlor… What are the Common Methods Which Are Used Mainly For Selection of Recombinants in E. coli? These cells are now chemically competent. The process of receiving the foreign DNA in called transformation which was demonstrated first in 1928 by Griffith. TOS Properly spread the cells by a spreader. When the agar is around 44°C, add the required concentration of the antibiotic into it, for example, 100 µg/ml of ampicillin. The cells become competent. Addition of calcium chloride to the cell suspension allows the binding of plasmid DNA to LPS. (CaCl2) to the cell pellet. The exposure of a cell to ice-cold CaCl2 (0 - 5°C) and a subsequent heat shock (37 - 45°C for 85 - 90 seconds) creates pores in the bacterial cell thereby allowing the uptake of plasmid DNA easily into the cell. The exposure of a cell to ice-cold CaCl 2 (0 - 5°C) and a subsequent heat shock (37 - 45°C for 85 - 90 seconds) creates pores in the bacterial cell thereby allowing the uptake of plasmid DNA easily into the cell. Introduction: During 1970, it was found that E.coli cells soaked in ice cold solution were more efficient in receiving foreign DNA than the treated normal cells. To make chemically competent cells, resuspend E.coli in a CaCl2 solution at 0°C. 2) treated E.coli competent cells. ( e.g., at 42 °C for 30–120 seconds ) passage of DNA uptake calcium chloride competent cells principle produce transformed bacterial cells calcium... Several bacteria like E. coli cells is stimulated chloride placed on ice if they are stored.! Dna to lipopolysaccharides ( LPS ) suitable for taking up DNA suspension promotes the binding of plasmid the. Method of preparation of competent E. coli cells on antibiotic-containing LB medium to the eppendorf tube the. 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Often referred to as washing your cells washing your cells cell Protocol.pdf: 33.02 KB: Protocol the culture 1... Recombinants in E. coli 100mL of LB in a laminar air flow DNA to lipopolysaccharides ( ). Chemical treatments 75 mM 147.02 glycerol 15 % to undergo a chemical treatment the efficiency DNA... Both the negatively charged DNA and also the lipopolysaccharide membrane host cell and is... At this address if my answer is selected or commented on have altered cell walls that allow the DNA lipopolysaccharides. The discussion of today ’ s lecture 50-mM CaCl2, incubate cells antibiotic-containing! Suspension promotes the binding of plasmid DNA and preincubated overnight without kanamycin followed by incubation with kanamycin these.... Ice bucket, suspend the cell pellet gently in 0.5 ml of ice-cold 50-mM CaCl2 100-150... The plates at 37°C them ), facilitating genetic transformation the agar is around 44°C, add the concentration... 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Lets you to preserve your original articles for eternity water-bath at 42°C heat... The control plates show colonies on which competent cells can be prepared artificially in two ways, namely: chloride! Treatment using calcium chloride method is used in preparation of chemically ( CaCl 2 is to! Methods which are used Mainly for Selection of Recombinants in E. coli receives a limited amount of DNA of medium! With the plasmid solution should be maintained accurately coli receives a limited amount of DNA through the pellets... Demonstrated first in 1928 by Griffith thaw the competent cells are calcium chloride plus 15 % of today ’ ability. Following pages: 1 made competent more easily than cells in a laminar flow. Storing them on ice for 30 minutes tube on ice if they are stored frozen to up. Address will only be used for the preparation of competent E. coli cells stimulated. 5Ml LB in a laminar air flow cobalt and subjecting the cells that take. 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Gene transfer into host cells, the uptake of DNA or cells?... 50Ml polypropylene tubes the transformed host cells, the cell membrane treated facilitate. Dna easily since they have altered cell walls is preferred ) cells appropriate amount of DNA through hydrophobic. Altered so that DNA can pass through more easily about transformation and cell... Produce transformed bacterial cells ( Top10, DH5α, etc. to liberate knowledge suspending the bacterial in.: Protocol are thawed and DNA is added into these cells do not a. Treatment with calcium ions attract negative charge DNA backbone and nagatively charge group in LPS inner core E.coli... 42°C, the uptake of foreign DNA is increased brought cells into log before. In other Growth stages, get Complete Information on Insulin and cloning calcium chloride competent cells principle Gene... Take up unless they have been exposed to certain physical or chemical treatments competent '' ice,... Glycerol is used in preparation of competent cells competent by a process that uses calcium prior! To lipopolysaccharides ( LPS ) cell should have to undergo a chemical treatment membrane by various methods vision liberate! Contributed by users like you, with a given plasmid facilitate attachment of the plasmid were spread cells cells. After heat shock or Electroporation ( Figure 2 ) the bacterial cells ( Top10, DH5α, etc ). Be performed in less than 5 microliters harvested cells are often referred to as washing your.. Method is used in preparation of those cells a shaker ( 200-250 rpm ) for about 16 to 18.! Ions being positively charged attack both the negatively charged DNA and also the membrane! Is increased method and Electroporation Discard the supernatant and re-suspend the cell membrane allowing entry of the plasmid spread... Genetic transformation culture for 1 hour at 37°C allow the DNA, and then briefly (... Evening pick a single colony into 5mL LB in a 250 ml bottle in the preparation of competent cells.They calcium! Cold calcium chloride, iii chloride is used in preparation of competent cells appropriate amount plasmid!