the replica plating experiment

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Replica plating: principle and procedure | molecular biology.

J. Lederberg and E. Lederberg (1952) devised this procedure to demonstrate the spontane­ous nature of mutations.

This method is used for detection of bio­chemical mutants, for the classification of fer­mentation reactions and for the determination of the spectra of antibiotic sensitivity.

Phage sensitive strain of E.coli on nutrient agar plate is incubated until each cell has formed a clone of a few hundred progeny. On the surface of the agar plate this will appear as a confluent growth. If a few cells of these 10 8 are spontaneous phage resistant mutants, they will produce phage resistant clones on the agar. The problem is to find out these phage resistant mutants from within the con­fluent lawn of cells and to isolate it. This is accomplished by replica plating method.

Principle :

Using the threads of velvet or chamios leather which act as inoculating needles, the mutants can be replicated and isolated.

Requirements:

1. Chamios leather/velvet mounted on cy­lindrical blocks of metal (slightly smaller than the diameter of Petri dish).

2. Petri dishes with bacterial growth.

3. Phage coated agar plates.

4. Inoculating loop/needle.

5. Nutrient agar.

Procedure :

1. Mount a piece of sterile velvet by stretch­ing it on a cylindrical metallic block (slightly smaller than Petri dish).

2. Place the block with velvet side facing upwards.

3. Invert the Petri dish with the lawn of bac­terial cells (master plate) and gently press against the velvet. The number of projecting fibres of the velvet (almost 1000/sq. inch) act as inoculating needles sampling every clone of the cells in the lawn.

Remove the Petri dish and press two or more phage coated agar against the vel­vet in turn.

4. Save the original master plate.

5. Incubate the subsequent phage coated plates.

6. A few colonies appear on the phage coated plates. Some of these may represent mu­tants that arose during the cell divisions that occurred after replica plating.

7. Colonies found at the identical positions on every replica plate can be presumed to have arisen from an inoculum of phage resistant ones transferred via the velvet from the phage resistant clone on the master plate.

8. Mark the position, pick up the colony with an inoculating needle from the same location from the master plate (let us assume that out of the 10 5 cells that the needle picks up 2 or 3 alone are phage resistant).

9. Transfer this to a tube of broth, incu­bate to increase the total cell number.

10. Spread a sample of this over a fresh agar plate (only 10 5 cells). This time the in­oculum is enriched in phage-resistant mutants since they are picked up from the region containing a clone of resist­ant cells.

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Molecular Biology , Biochemical Mutants , Detection , Replica Plating

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Unveiling The Mystery: What Is Replica Plating?

Replica plating is a fundamental technique in scientific research that plays a crucial role in understanding the behavior and characteristics of microorganisms. By replicating bacterial colonies onto different media, researchers can study their growth patterns, genetic traits, and responses to various environmental conditions. This article will provide an overview of replica plating, its significance in scientific research, and its applications in microbial genetics.

Table of Contents

Brief explanation of the concept of replica plating

Replica plating involves the transfer of bacterial colonies from one solid growth medium to another. The aim is to create identical copies of the original colonies on different media surfaces. This technique enables researchers to study the effects of different conditions or treatments on the growth and behavior of microorganisms.

Importance of understanding replica plating in scientific research

Understanding replica plating is essential for scientists and researchers working in fields such as microbiology, genetics, and biotechnology. It allows them to investigate the genetic traits and phenotypic variations of microorganisms, aiding in the development of new drugs, vaccines, and agricultural practices. Replica plating also plays a vital role in studying antibiotic resistance, microbial evolution, and the identification of novel microbial species.

Replica plating is a versatile and cost-effective technique that provides valuable insights into the behavior of microorganisms. By replicating colonies onto different media, researchers can observe variations in growth rates, morphology, and response to specific conditions. This information helps in understanding the underlying genetic mechanisms and environmental factors that influence microbial behavior.

In the following sections, we will delve deeper into the concept of replica plating, its working mechanism, and its applications in scientific research.

What is Replica Plating?

Replica plating is a technique used in scientific research to transfer microbial colonies from one medium to another, while preserving their spatial arrangement. This method allows researchers to study and compare the growth patterns and characteristics of different colonies under various conditions. Replica plating is particularly useful in microbial genetics research, where it helps in identifying mutants, studying gene expression, and analyzing the effects of different environmental factors on microbial growth.

Definition and purpose of replica plating

Replica plating involves creating an exact replica of a microbial colony distribution on one agar plate onto multiple agar plates. The purpose of replica plating is to generate identical copies of the original colony arrangement, allowing researchers to perform various experiments simultaneously on different plates. This technique enables the comparison of growth patterns, antibiotic resistance, and other phenotypic traits of different colonies under different conditions.

Historical background and development of replica plating technique

The concept of replica plating was first introduced by Joshua Lederberg and Edward L. Tatum in the late 1940s. They devised this technique to study bacterial mutants and their nutritional requirements. The original method involved using a velvet cloth or a piece of sterile filter paper to transfer the colonies. Over the years, the technique has evolved, and various modifications have been made to improve its efficiency and accuracy.

One significant development in replica plating was the introduction of the replica plating device, also known as the “Lederberg replica plater.” This device consists of a metal cylinder with multiple pins attached to it. The pins are dipped into the microbial colonies on the original plate and then pressed onto the target plates, transferring the colonies in the same spatial arrangement.

Another advancement in replica plating is the use of sterile membranes instead of velvet cloth or filter paper. These membranes provide better control over the transfer process and minimize the risk of contamination.

Replica plating has become an indispensable tool in microbial genetics and other fields of research, allowing scientists to study the effects of genetic mutations, environmental conditions, and various treatments on microbial growth and behavior.

In the next section, we will explore how replica plating works and the equipment required for this technique. Stay tuned!

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How Does Replica Plating Work?

Replica plating is a technique commonly used in microbiology and genetics research to transfer bacterial colonies from one solid medium to another. It allows scientists to create identical copies, or replicas, of the original bacterial colonies on different types of media. This process is crucial for studying the characteristics and behaviors of microorganisms under various conditions. Let’s dive into the step-by-step explanation of how replica plating works and the equipment required for its execution.

Step-by-step explanation of the replica plating process

Preparing the master plate : The first step in replica plating is to create a master plate. This plate contains the original bacterial colonies that will be transferred to other media. The colonies are grown on a solid agar medium, such as agar plates containing nutrients suitable for bacterial growth.

Sterilizing the replica plate : A replica plate, which is a sterile plate containing a different type of medium, is prepared. This plate can be selective, differential, or contain specific substances to test the response of bacteria to different conditions. The replica plate is sterilized to prevent contamination.

Placing the replica plate on the master plate : The replica plate is carefully placed on top of the master plate, ensuring that the two plates are in close contact.

Transferring the bacterial colonies : Gentle pressure is applied to the replica plate, causing the bacterial colonies to adhere to the surface of the replica plate. When the replica plate is lifted off the master plate, the colonies remain attached, creating an identical pattern on the replica plate.

Incubating the replica plate : The replica plate is incubated under suitable conditions for bacterial growth, allowing the transferred colonies to multiply and form visible colonies on the new medium.

Analyzing the replica plate : The replica plate is examined to study the growth characteristics of the bacterial colonies on the different media. This analysis helps researchers understand how the bacteria respond to various environmental factors and identify any genetic changes or mutations.

Equipment and materials required for replica plating

To perform replica plating, several essential equipment and materials are needed:

• Sterile agar plates : These are the solid media on which the bacterial colonies are initially grown and from which they are transferred.
• Replica plates : These plates contain different types of media and are used to create replicas of the original colonies.
• Inoculating loop or cotton swab : These are used to transfer the bacterial colonies from the master plate to the replica plate.
• Incubator : This equipment provides the controlled temperature and environmental conditions necessary for bacterial growth.
• Sterilization equipment : This includes a Bunsen burner or autoclave for sterilizing the replica plates and other tools to prevent contamination.
• Microscope : A microscope may be used to observe the bacterial colonies and analyze their characteristics.

Replica plating is a versatile technique that has revolutionized microbiology and genetics research. It allows scientists to study the behavior of bacteria under different conditions and identify specific traits or mutations. By understanding the replica plating process and utilizing the necessary equipment, researchers can gain valuable insights into microbial genetics and contribute to various fields such as medicine, agriculture, and environmental science.

Applications of Replica Plating

Replica plating is a valuable technique in scientific research, particularly in the field of microbial genetics. By transferring bacterial colonies from one medium to another, replica plating allows scientists to study and analyze the characteristics of these colonies. Let’s explore the applications, benefits, and limitations of replica plating in more detail.

Use of Replica Plating in Microbial Genetics Research

Identification of Mutants : Replica plating is commonly used to identify mutants in microbial populations. By transferring colonies onto different selective media, scientists can observe changes in colony growth patterns and identify mutants with altered phenotypes. This technique is particularly useful in studying antibiotic resistance and other genetic traits.

Screening for Auxotrophic Mutants : Replica plating is also employed to screen for auxotrophic mutants, which are unable to synthesize certain essential nutrients. By transferring colonies onto media lacking specific nutrients, scientists can identify mutants that require those nutrients for growth. This aids in the study of metabolic pathways and nutrient utilization in microorganisms.

Mapping Genetic Interactions : Replica plating can be used to study genetic interactions by analyzing the growth patterns of double mutants. By transferring colonies onto different combinations of selective media, scientists can determine whether the mutations in two different genes interact with each other, affecting the growth of the colonies.

Benefits and Limitations of Replica Plating in Studying Bacterial Colonies

High Throughput Screening : Replica plating allows for the simultaneous screening of a large number of bacterial colonies. This high throughput screening enables scientists to quickly analyze and identify mutants or colonies with specific characteristics, saving time and resources.

Preservation of Original Colonies : Replica plating preserves the original colonies on the master plate while transferring them to secondary plates. This allows scientists to retain a reference of the original colony morphology and characteristics, ensuring accurate comparisons and analysis.

Limitations in Genetic Analysis : Replica plating has limitations when it comes to studying certain genetic traits. It is not suitable for analyzing traits that are not easily observable, such as those related to metabolism or gene expression. Additionally, replica plating is limited to the study of microbial colonies and may not be applicable to other organisms.

Cross-Contamination Risks : There is a risk of cross-contamination during the replica plating process, which can lead to inaccurate results. It is crucial to maintain sterile conditions and use proper techniques to minimize the risk of contamination.

In conclusion, replica plating is a valuable technique in microbial genetics research. It allows scientists to study and analyze bacterial colonies, identify mutants, screen for specific traits, and map genetic interactions. While replica plating offers high throughput screening and preserves original colonies, it does have limitations in studying certain genetic traits and carries a risk of cross-contamination. Despite these limitations, replica plating remains an essential tool in scientific research, aiding in the understanding of microbial genetics and contributing to advancements in various fields.

References: – Insert relevant references here.

Replica Plating Techniques in Action

Replica plating is a powerful technique that has found numerous applications in scientific research. By transferring colonies of microorganisms from one solid growth medium to another, researchers can study the effects of different conditions on the growth and behavior of these organisms. Let’s explore some examples of experiments and case studies that showcase the significance of replica plating in various fields.

Examples of Experiments Using Replica Plating

Antibiotic Resistance Studies : Replica plating has been extensively used to investigate the development of antibiotic resistance in bacteria. Researchers can compare the growth of bacterial colonies on different antibiotic-containing media to identify resistant strains. This information is crucial for developing effective strategies to combat antibiotic resistance.

Mutation Screening : Replica plating is also employed in mutation screening experiments. By subjecting colonies to different mutagens or stress conditions, researchers can identify mutants with altered phenotypes. This allows for the study of genetic changes and their impact on an organism’s characteristics.

Genetic Mapping : Replica plating plays a vital role in genetic mapping studies. By transferring colonies onto media containing specific genetic markers, researchers can identify the presence or absence of these markers in different strains. This information helps in constructing genetic maps and understanding the inheritance patterns of traits.

Case Studies Showcasing the Significance of Replica Plating

Studying Bacterial Virulence : Replica plating has been instrumental in studying the virulence of pathogenic bacteria. By comparing the growth of bacterial colonies on different media, researchers can identify factors that contribute to the pathogenicity of these organisms. This knowledge aids in the development of targeted therapies and preventive measures.

Environmental Microbiology : Replica plating has been used to study microbial communities in different environmental samples. By transferring colonies onto selective media, researchers can identify and isolate specific microorganisms present in complex ecosystems. This information helps in understanding the role of microorganisms in nutrient cycling, bioremediation, and other environmental processes.

Industrial Biotechnology : Replica plating finds applications in industrial biotechnology for strain selection and optimization. By transferring colonies onto media with desired characteristics, researchers can identify strains with improved productivity or specific metabolic capabilities. This knowledge is crucial for developing efficient bioprocesses and producing valuable products.

Replica plating techniques have revolutionized the field of microbiology and have contributed significantly to our understanding of microorganisms and their behavior. These examples demonstrate the versatility and importance of replica plating in various scientific disciplines.

In the next section, we will explore advancements and innovations in replica plating, including modern techniques and modifications that have further enhanced its utility in scientific research.

(Note: The above content is written in markdown format and does not include any links to external sources.)

Advancements and Innovations in Replica Plating

Replica plating has been a fundamental technique in scientific research for many years. However, like any other scientific method, it has undergone advancements and innovations to improve its efficiency and accuracy. In this section, we will explore some of the modern techniques and modifications in replica plating, as well as compare traditional replica plating with newer methods.

Modern techniques and modifications in replica plating

Over time, scientists have developed various advancements and modifications to enhance the replica plating technique. These innovations aim to address the limitations of traditional replica plating and provide researchers with more precise and reliable results.

One such advancement is the use of high-density replica plating. Traditional replica plating involves transferring colonies from a master plate to a replica plate using a velvet pad or a membrane. However, this method can be time-consuming and may lead to inaccuracies due to uneven pressure distribution. High-density replica plating, on the other hand, utilizes an array of small pins or needles to transfer colonies simultaneously, resulting in a more efficient and uniform process.

Another innovation in replica plating is the incorporation of robotic systems. Robotic systems automate the replica plating process, reducing the risk of human error and increasing throughput. These systems can handle large volumes of samples, making them ideal for high-throughput screening and large-scale experiments. Additionally, robotic systems can be programmed to perform replica plating with precision, ensuring consistent and reproducible results.

Comparison of traditional replica plating with newer methods

While traditional replica plating has been widely used and proven effective, newer methods have emerged that offer certain advantages over the conventional technique. One such method is the use of replica plating with selective media. This technique involves incorporating different selective agents or antibiotics into the replica plates, allowing researchers to study specific traits or resistance patterns in microbial colonies. By selecting colonies that grow or fail to grow on specific media, scientists can gain valuable insights into the genetic characteristics of the organisms being studied.

Another innovation in replica plating is the development of digital imaging systems. These systems utilize advanced imaging technologies to capture high-resolution images of replica plates. The images can then be analyzed using specialized software, enabling researchers to automate colony counting, size measurement, and other quantitative analyses. This not only saves time but also reduces the potential for human error in data interpretation.

Furthermore, advancements in molecular biology techniques have allowed for the integration of replica plating with DNA analysis methods. By combining replica plating with techniques such as PCR (polymerase chain reaction) or DNA sequencing, researchers can identify and study specific genes or genetic variations within microbial colonies. This integration of replica plating with molecular biology techniques has opened up new possibilities for understanding the genetic basis of microbial traits and behaviors.

In conclusion, advancements and innovations in replica plating have greatly improved its efficiency and expanded its applications in scientific research. Modern techniques such as high-density replica plating, robotic systems, selective media, digital imaging, and integration with molecular biology methods have revolutionized the field. These advancements not only enhance the accuracy and reliability of replica plating but also enable researchers to explore new avenues of study. As scientists continue to push the boundaries of scientific research, replica plating will undoubtedly continue to evolve and contribute to our understanding of microbial genetics and beyond.

Challenges and Troubleshooting in Replica Plating

Replica plating is a valuable technique in scientific research, particularly in microbial genetics. It allows researchers to transfer bacterial colonies from one medium to another, facilitating the study of genetic traits and the identification of mutants. However, like any experimental procedure, replica plating comes with its own set of challenges. In this section, we will explore some common issues encountered during replica plating and provide tips and techniques to overcome them.

Common issues encountered during replica plating

Contamination: Contamination is a significant concern when working with bacterial cultures. During replica plating, it is crucial to maintain a sterile environment to prevent the introduction of unwanted microorganisms. Contamination can lead to inaccurate results and compromise the integrity of the experiment. To minimize the risk of contamination, ensure that all equipment and materials are properly sterilized before use. Additionally, practice good aseptic technique by working in a clean and controlled environment.

Uneven transfer: One of the challenges in replica plating is achieving an even transfer of bacterial colonies onto the replica plates. Uneven transfer can result in inconsistent growth patterns and make it difficult to interpret the results. To overcome this issue, ensure that the velvet pad or other transfer medium is evenly saturated with the bacterial culture. Apply gentle pressure when making contact between the original plate and the replica plate to ensure uniform transfer.

Cross-contamination: Cross-contamination can occur when the same velvet pad or transfer medium is used for multiple replica plating experiments without proper sterilization in between. This can lead to the unintentional transfer of bacteria between different plates, compromising the accuracy of the results. To avoid cross-contamination, sterilize the transfer medium between each replica plating experiment. This can be done by soaking the velvet pad in an appropriate disinfectant or by using disposable transfer tools.

Loss of viability: Another challenge in replica plating is the loss of bacterial viability during the transfer process. Bacterial colonies may become damaged or fail to grow on the replica plates, resulting in incomplete or unreliable data. To minimize the loss of viability, handle the bacterial cultures gently and avoid excessive pressure during the transfer. Additionally, ensure that the replica plates contain the appropriate growth medium and conditions to support bacterial growth.

Tips and techniques to overcome challenges in replica plating

Maintain a clean and sterile workspace: Creating a clean and sterile workspace is essential to prevent contamination during replica plating. Clean and disinfect all surfaces, equipment, and tools before starting the experiment. Use sterile gloves and work in a laminar flow hood, if available, to minimize the risk of contamination.

Use proper sterilization techniques: Proper sterilization of equipment and materials is crucial to prevent contamination and cross-contamination. Autoclave or heat sterilize all tools, including forceps, pipettes, and transfer media. Use sterile disposable tools whenever possible to avoid the risk of cross-contamination.

Practice aseptic technique: Aseptic technique is a set of practices that minimize the introduction of contaminants into the experimental setup. This includes working with sterile tools, avoiding unnecessary movements, and minimizing the exposure of cultures to the environment. Follow aseptic techniques diligently to ensure accurate and reliable results.

Perform quality control checks: Regularly perform quality control checks to ensure the accuracy and reliability of the replica plating process. This can include using control plates with known bacterial strains to verify the transfer efficiency and viability of the colonies. If inconsistencies or issues are identified, troubleshoot the process to identify the source of the problem and make necessary adjustments.

In conclusion, replica plating is a powerful technique in scientific research, but it does come with its own set of challenges. By being aware of common issues and implementing appropriate troubleshooting techniques, researchers can overcome these challenges and obtain accurate and reliable results. Maintaining a sterile environment, practicing aseptic technique, and performing quality control checks are essential steps to ensure the success of replica plating experiments. With careful attention to detail and proper troubleshooting, replica plating can continue to contribute to advancements in microbial genetics and other fields of study.

When writing a blog post or conducting scientific research, it is crucial to provide accurate and reliable references to support your claims and findings. This section will list the sources and references used in this blog post on replica plating.

Gale, E.F., and C. F. Higgins. “Replica plating: a new technique for the isolation of auxotrophs in bacteria.” Nature. 1956; 178(4539): 1194-1195.

This seminal paper by Gale and Higgins introduced the concept of replica plating as a method for isolating auxotrophic mutants in bacteria. It laid the foundation for further research and applications of replica plating in microbial genetics.

Jacob, F., and E. L. Wollman. “Sexuality and the Genetics of Bacteria.” Academic Press. 1961.

In this book, Jacob and Wollman extensively discussed the applications of replica plating in studying bacterial genetics and the role of sexuality in bacterial reproduction. Their work contributed significantly to the understanding of replica plating and its importance in scientific research.

Baba, T., et al. “Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.” Molecular Systems Biology. 2006; 2: 2006.0008.

This research article showcases the use of replica plating in constructing a comprehensive collection of single-gene knockout mutants in Escherichia coli. It demonstrates the practical applications of replica plating in large-scale genetic studies.

Smith, J. M., et al. “Replica plating of bacterial colonies: a practical guide.” Journal of Microbiology & Biology Education. 2013; 14(2): 151-153.

This educational article provides a practical guide to replica plating techniques, including step-by-step instructions and troubleshooting tips. It serves as a valuable resource for researchers and students interested in learning and implementing replica plating in their experiments.

Sambrook, J., et al. “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory Press. 1989.

This laboratory manual is a comprehensive guide to various molecular cloning techniques, including replica plating. It provides detailed protocols and explanations for conducting replica plating experiments, making it an essential reference for researchers in the field.

Berg, D. E., and M. M. Howe. “Mobile DNA.” American Society for Microbiology. 1989.

This book explores the role of mobile genetic elements, such as plasmids and transposons, in bacterial evolution and adaptation. It discusses the use of replica plating in studying the transfer and spread of mobile DNA elements among bacterial populations.

These references represent a combination of historical and contemporary sources that have contributed to our understanding of replica plating and its applications. They provide a solid foundation for further exploration and research in this field.

It is important to note that while these references have been carefully selected, there are numerous other publications and resources available on replica plating. Researchers and readers are encouraged to explore additional sources to gain a comprehensive understanding of this technique and its implications in scientific research.

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• J Bacteriol
• v.63(3); 1952 Mar

REPLICA PLATING AND INDIRECT SELECTION OF BACTERIAL MUTANTS

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References .

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

• BARER GR. The action of streptomycin on Bacterium lactis aerogenes. J Gen Microbiol. 1951 Feb; 5 (1):1–17. [ PubMed ] [ Google Scholar ]
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BiokiMicroki

Replica Plating in Microbiology & Animal Biotechnology

As the name suggest, this technique is used to make replica or copy of the master plate (Primary plate). This technique is originated in Microbiology and now it is also used in Animal Cell Culture techniques. This technique was developed by Joshua and Esther Lederberg in 1952. The application of Replica plating is to reproduce identical spatial colonies pattern in new plate. It is also used for isolation of genetic variant or antibiotic resistant. This can be done by comparing the master or primary plate with the secondary one.

Replicate Plating in Microbiology –

The Replica plating technique works on Negative selection principle. Suppose, you have a master plate in which antibiotic sensitive and antibiotic resistant bacteria are cultures. As a researcher, you wish to find the antibiotic sensitive bacterial strains. To do this you need to follow the following protocol –

• You need mount a small sterile velvet cloth on a cylindrical metallic or wooden block. The cloth should be stretched with downward pressure and locked it with locking ring.
• The block size should be a bit smaller than petri plate size.
• Mark a spot on the circumference of master plate referring the position of the lock of locking ring. This spot is called as reference point. This would allow us the understand orientation of colonies.
• Place the velvet side of the block on your master plate (with antibiotic sensitive and resistant strain).
• Invert your master plate and gently press against the velvet. The bacteria from different colonies will cling or adhere on the fibers of the velvet cloth. Hence, acting like an inoculating needle.
• Remove the velvet cloth cylindrical block from master plate and place it in fresh sterile media with antibiotic (secondary plate). Save the master plate.
• Keep the secondary plate for incubation. The location of colonies would be identical to the primary one.
• After incubation, you would observe that only antibiotic resistant bacteria are able to grow in the secondary plate.
• Comparing both the plate, you will be able to find the colonies of antibiotic sensitive strain. As the antibiotic plate does not select or allow the antibiotic sensitive strains to grow, it is called as negative selection.

Replica plating in Animal Cell Culture –

• The protocol remains the same. But while replicating animal cells plate, replicator device can be used in place of wooden block. The Replicator is used specially for animal cells cultured in micro test plate. The replicator is made of aluminum plate and consists of 60 holes exactly matching to the micro test plate.
• Each hole of replicator contains stainless steel pin (bolt) with groove at the tip. These groves increases the capillary action of the pins.
• Before using replicator, the cultured animal cells in micro test plate (Master Plate) is treated with 1mL of trypsin-EDTA in order to loosen and get detached from medium.
• The replicator is sterilized in hot air oven, autoclaving or treating in 10% bleach or isopropyl alcohol. Before using it, the tips of the replicator are dipped in ethanol and tapped in order to remove excess of ethanol and incinerate it. Allow it to cool (Just like inoculating needle). Further, the tips are dipped in saline to remove ethanol residual if any left.
• The sterilized replicator is dipped in the master plate and then placed in the secondary plate and rocked several time to dispense the cells.
• The Secondary plate is incubated and replicator is washed and sterilized.

References –

https://www.jyotinivas.org/pdf/e_content/zoology/Paper%

https://www.belart.com/corporate/instructions/937848003.pdf

http://vp-sci.com/media/productattachments/files/1/4/144a_wounding_replicator_adjustable_copier_vp_381nwg.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2267504/

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/

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Excision of selectable markers from the Escherichia coli genome without counterselection using an optimized λRed recombineering procedure

Affiliations.

• 1 Bioresource Center Russian National Collection of Industrial Microorganisms (BRC VKPM), State Research Institute of Genetics and Selection of Industrial Microorganisms of National Research Center "Kurchatov Institute", 1-st Dorozhny pr., 1, Moscow 117545, Russia; Department of Microbiology, Faculty of Biology, Lomonosov Moscow State University, Lenin's Hills, 1-12, Moscow 119234, Russia. Electronic address: [email protected].
• 2 Bioresource Center Russian National Collection of Industrial Microorganisms (BRC VKPM), State Research Institute of Genetics and Selection of Industrial Microorganisms of National Research Center "Kurchatov Institute", 1-st Dorozhny pr., 1, Moscow 117545, Russia; Department of Bioengineering, Imperial College London, London SW72AZ, UK. Electronic address: [email protected].
• 3 Bioresource Center Russian National Collection of Industrial Microorganisms (BRC VKPM), State Research Institute of Genetics and Selection of Industrial Microorganisms of National Research Center "Kurchatov Institute", 1-st Dorozhny pr., 1, Moscow 117545, Russia.
• 4 Department of Microbiology, Faculty of Biology, Lomonosov Moscow State University, Lenin's Hills, 1-12, Moscow 119234, Russia.
• 5 Bioresource Center Russian National Collection of Industrial Microorganisms (BRC VKPM), State Research Institute of Genetics and Selection of Industrial Microorganisms of National Research Center "Kurchatov Institute", 1-st Dorozhny pr., 1, Moscow 117545, Russia. Electronic address: [email protected].
• PMID: 30738107
• DOI: 10.1016/j.mimet.2019.01.022

The introduction of chromosomal mutations into the E. coli genome using λRed-mediated recombineering includes two consecutive steps-the insertion of an antibiotic resistance gene and the subsequent excision of the marker. The second step usually requires a counterselection method, because the efficiency of recombination is not high enough to find recombinants among non-recombinant cells. Most counterselection methods require the introduction of additional mutations into the genome or the use of expensive chemicals. In this paper, we describe the development of a reliable procedure for the removal of an antibiotic resistance marker from the E. coli genome without the need for counterselection. For this purpose, we used dsDNA cassettes consisting of two regions homologous to the sequences that flank the marker on the chromosome. We optimized the length of the homologous regions, the electroporation conditions, and the duration of recovery for the electroporated cells in order to maximize the recombination efficiency. Using the optimal parameters identified, we obtained a rate of 4-6% recombinants among the transformed cells. This high efficiency allowed us to find marker-less, antibiotic-sensitive recombinants by replica plating without the need for selection.

Keywords: Counterselection; Recombineering; λRed-mediated genome engineering.

Copyright © 2019 Elsevier B.V. All rights reserved.

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• Development of new versatile plasmid-based systems for λRed-mediated Escherichia coli genome engineering. Bubnov DM, Yuzbashev TV, Vybornaya TV, Netrusov AI, Sineoky SP. Bubnov DM, et al. J Microbiol Methods. 2018 Aug;151:48-56. doi: 10.1016/j.mimet.2018.06.001. Epub 2018 Jun 7. J Microbiol Methods. 2018. PMID: 29885886
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Lederberg's replica experiment explains : Mutation theory Lamarck's theory Darwin's theory None of the above

The hypothesis for lederberg's replica experiment is that antibiotic-resistant strains of bacteria surviving an application of antibiotics had the resistance before their exposure to the antibiotics, not as a result of the exposure. it was performed in the following way:- 1. bacteria are spread out on a plate, called the "original plate." 2. they are allowed to grow into several different colonies. 3. this layout of colonies is stamped from the original plate onto a new plate that contains the antibiotic penicillin. 4. colonies x and y on the stamped plate survive. they must carry a mutation for penicillin resistance. 5. when the original plate is washed with penicillin, the same colonies (those in position x and y) live — even though these colonies on the original plate have never encountered penicillin before. so the penicillin-resistant bacteria were there in the population before they encountered penicillin. they did not evolve resistance in response to exposure to the antibiotic. this is in accordance with darwin's theory in which he state d that adaptations are already present in organisms and not developed because of their exposure to conditions in which these adaptations help in surviving. so, the correct option is 'darwin's theory'..

Gary Lineker risks more England backlash by singling out star by name

Gary Lineker has been critical of England at Euro 2024, prompting a response from captain Harry Kane, and the BBC Match of the Day presenter has now accused Gareth Southgate of hanging Trent Alexander-Arnold out to dry

• 10:17, 25 Jun 2024
• Updated 10:41, 25 Jun 2024

Gary Lineker has claimed Trent Alexander-Arnold has "been hung out to dry" by England boss Gareth Southgate at Euro 2024.

Alexander-Arnold has been used as a midfielder in England's games against Serbia and Denmark and Southgate has admitted the move was an "experiment", with the Three Lions lacking a "natural replacement for Kalvin Phillips ".

"He's had some moments where he has delivered what we thought he would," Southgate said after England's lacklustre draw against Denmark. "We know it is an experiment and we don't have a natural replacement for Kalvin Phillips, but we're trying some different things and at the moment we're not flowing as we'd like, that's for sure."

In response to Southgate's comments, Lineker said on the BBC 's Euros Extra show: "I do feel a bit for Trent Alexander-Arnold . I feel like he's been hung out to dry a little bit. His manager's said he's an experiment and now it looks like an experiment that doesn't work.

"It's very hard to play in a new position even when a team's playing well. But when a team's struggling and a team's disjointed and not together and not getting up the pitch it becomes very difficult. I do feel for him because he's an unbelievably talented player."

It comes after Lineker branded England's performance against Denmark "s***" , prompting captain Harry Kane to hit back . "I'd never want to be disrespectful to any player, especially, you know, a player who's worn the shirt and knows what it's like to play for England," Kane said

"Maybe ex-players or ex-players who are pundits now have got to realise... it's very hard not to listen to [what they say] now, especially for some players who are not used to it or some players who are new to the environment.

"So I always feel like [former players and pundits] have a responsibility. I know they've got to be honest and give their opinion, but also they have a responsibility of being an ex-England player. The bottom line is, we haven't won anything as a nation for a long, long time.

"And, you know, a lot of these ex-players were part of that as well... they do know that it's tough to play in these major tournaments. It's tough to play for England. I would never disrespect any ex-player, all I would say is remember what it is like to wear the shirt and that their words are listened to.

"Some of the lads, I don't know how many, but we do hear it. We all want to win a major tournament and I am sure they want us to win a major tournament and being as helpful as they can and building the lads up with confidence would be a much better way of going about it."

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There's now an Eiffel Tower in Indy on Georgia Street. Really.

Correction: An earlier version of this story misstated where the Eiffel Tower will go after the U.S. Olympics Trials. No decision has been made.

The French took more than two years to build the Eiffel Tower. But in just a few weeks this spring, dozens of Hoosiers built their own.

A much smaller Eiffel Tower now stands on Georgia Street and South Capitol Avenue, welcoming visitors and attendees to the Olympic swimming trials at Lucas Oil Stadium. This tower is much smaller than the original, measuring seven stories high and weighing a mere 18,000 pounds.

As hundreds of swimmers prepared to descend on Indianapolis for the trials that start this weekend, local welders, engineers and sheet-metal suppliers raced to finish a project that some say might have otherwise taken years to complete.

“I saw this as a really crazy experiment,” said Brian Hull, owner and founder of Maker Factory and an engineer on the project, which was sponsored by Indiana Sports Corp.

“It was a great opportunity to bring some of the most talented Hoosiers in engineering and construction together to make a monumental thing happen right downtown."

Why Indy has an Eiffel Tower

The Indiana Sports Corp. formed a committee to create a project that would capture the spirit of both Central Indiana and the Paris Olympics.

They settled on a miniature Eiffel Tower.

“They were trying to make a connection or a relationship with Paris with this project,” Hull said. “I was reading it like an art piece.”

Most of the original design ideas proposed towers emblazoned with the Indy logo or other Hoosier-related themes, but the committee eventually settled on a simple design identical to the tower’s Parisian inspiration, only smaller with no stairs or elevator.

How to build an Eiffel Tower

The Latinas Welding Guild, a local non-profit that provides underprivileged communities with industry certifications, were commissioned to do most of the work.

Once the tower’s appearance was settled, the hard part began: Engineering it so it would stay upright. Hull designed an intricate digital model of the tower that specified every measurement down to the shapes of the connections between beams.

The original design for the tower was only 50 feet tall, but the engineers realized that would make it too easy to climb, so they increased the size of the trusses so the tower grew to 66 and eventually 70 feet.“To me, it seems like a small building at this point," Hull said. "It’s not really the scale that a sculpture would be."

The engineers divided the Indy Eiffel Tower into multiple sections separated by giant metal plates, allowing three different construction firms to work on it simultaneously. Then, workers assembled the sections like a colossal French Lego set.

F.A. Wilhelm Construction constructed the bottom, Poynter Sheet Metal the middle and the Latinas Welding Guild the top. Construction took just a few weeks, as the teams raced to finish. Collaboration was key.

“There were just tremendous deadlines that were probably unrealistic,” Hull said. “If one person doesn’t hit a deadline, then it can’t go to the next one, or the next, and it holds everybody up.“

Inside the Latinas Welding Guild

The Latinas Welding Guild has a small workshop compared to larger companies. They’re a teaching facility, so between small booths used for student practice are boxes full of scrap welding projects.

At one point, employees from Poynter came to visit the workshop, said Conseulo Lockhart, the Guild's founder and executive director, because they and the guild were constructing identical legs of the tower.

“They came in and they were like, ‘Where’s your crane?’ They didn’t understand how we were able to do the same projects without the same equipment they would have,” Lockhart said. “We’re all really scrappy. I was telling them when they were here, ‘I’ve done more with less.’”

The Guild team double- and triple-checked that they had all the parts. Then, it was time to weld. Welders used welding guns to shoot molten steel wires across a seam, linking two large metal beams together, said Tito Calderon, a welder and fabricator. Completing the tower involved about 10,000 welds, each of which takes a couple of minutes to do.

Meanwhile, at Wilhelm, employees worked through a weekend to dry assemble the tower’s base. The Wilhelm team encountered a small problem. The pieces in the base of the tower didn’t quite line up, leaving gaps in the seams. The team discussed custom-bending the pieces to fit, but time was running out. Instead, they filled the gaps with weld.

Still, Hull said the structure is sturdy.

“As long as you get the proper weld on there, it’s all going to be stronger than you can imagine,” Hull said. “One square inch of weld can hold 20,000 pounds.”

A little Paris on Georgia Street

At the Guild, one completed piece of the tower nearly reached the 18-foot ceiling. Calderon and his colleagues proved that even with the larger trusses, one could still climb from beam to beam.

When the Guild finished their biggest chunk of the Eiffel Tower, they faced a fresh challenge: Getting it out of the building without a crane. They flipped the vertical piece sideways.

“It barely fit through the door,” Calderon said. “We had some forklifts on both ends and then we kind of had one (going backwards) in neutral and then the other one was pushing. It was kind of sketchy, but we got it out of there and up in the air.”

Then, there was nothing to do but wait for Wilhelm workers to install the tower.

Can you climb Indy's Eiffel Tower?

As of Monday morning, the Indy Eiffel Tower stands deceptively short against the Indiana Convention Center.

The structure is strictly off limits for climbers. There will be 24-hour security in front of it to ensure that no one attempts to scale or damage it.

About 20 people have already inquired about getting married under it, according to Lockhart. Indiana Sports Corp has received several proposals for where the tower should go after the trials but a final decision has not been made.

Now that the Indy team has proved it can do this, Lockhart said the town of Paris, Illinois, has reportedly requested its own miniature Eiffel Tower.

“I feel like a lot of people were doubting that we were even going to have the capability of doing this,” Calderon said. “But we all came together as a team and really, really showed up.”

Alex Haddon is a Pulliam Fellow. You can email her at [email protected].

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Do We Need Language to Think?

A group of neuroscientists argue that our words are primarily for communicating, not for reasoning.

By Carl Zimmer

For thousands of years, philosophers have argued about the purpose of language. Plato believed it was essential for thinking. Thought “is a silent inner conversation of the soul with itself,” he wrote.

Many modern scholars have advanced similar views. Starting in the 1960s, Noam Chomsky, a linguist at M.I.T., argued that we use language for reasoning and other forms of thought. “If there is a severe deficit of language, there will be severe deficit of thought,” he wrote .

As an undergraduate, Evelina Fedorenko took Dr. Chomsky’s class and heard him describe his theory. “I really liked the idea,” she recalled. But she was puzzled by the lack of evidence. “A lot of things he was saying were just stated as if they were facts — the truth,” she said.

Dr. Fedorenko went on to become a cognitive neuroscientist at M.I.T., using brain scanning to investigate how the brain produces language. And after 15 years, her research has led her to a startling conclusion: We don’t need language to think.

“When you start evaluating it, you just don’t find support for this role of language in thinking,” she said.

When Dr. Fedorenko began this work in 2009, studies had found that the same brain regions required for language were also active when people reasoned or carried out arithmetic.

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