DNA Extraction Lab Notes
Lab Objectives
This laboratory focuses on extracting plasmid DNA from E. coli and high molecular weight (HMW) DNA from fruits, specifically bananas. The following objectives guide the activities:
Extraction Techniques: Utilize the molecular properties of DNA to carry out DNA extractions.
Mini Plasmid Prep: Conduct a mini plasmid preparation to purify plasmid DNA for use in subsequent labs, specifically in transforming competent cells.
Protocol Design: Apply learned concepts to design a protocol for high molecular weight DNA extraction from a banana.
Equipment Utilization: Learn to use standard laboratory equipment such as micropipettors, microcentrifuges, and aseptic techniques effectively.
Background & Importance
DNA is crucial as it carries the genetic information necessary for the cell’s functioning. Understanding DNA extraction is pivotal in molecular biology for various applications, including genetic engineering and studies on antibiotic resistance. This lab enables students to practically engage with live cultures, fostering understanding of the methods and techniques used in genetic manipulation and molecular biology research.
Types of DNA
Prokaryotic DNA
Bacterial Genomic DNA:
Encodes essential functions for survival with approximately 4000 genes. Arranged in a circular form anchored to the cell membrane.
Bacterial Plasmid DNA:
Smaller, circular, and often supercoiled, containing about 2-25 genes. These plasmids can convey advantages, such as antibiotic resistance, but are not essential for basic survival.
Eukaryotic DNA
Organized into linear chromosomes located in the nucleus. Humans possess 23 pairs of chromosomes, with a total of approximately 3.2 billion base pairs. The extraction of eukaryotic DNA differs, particularly in terms of protocols and desired purity due to its complexity.
Understanding Plasmid Extraction Steps
Overall Procedure
The plasmid extraction involves multiple steps:
Culture Preparation:
Transfer E. coli from liquid culture into microcentrifuge tubes and centrifuge to pellet cells.
Resuspension:
Use STE (sterile TE buffer) to rinse and resuspend the bacterial pellet.
Lysis of Cells:
Add Solution I (isotonic solution) to maintain osmotic balance during the re-suspension of cells.
Add Solution II (detergent and sodium hydroxide) to lyse cells, denature DNA, and release cellular components.
Neutralization:
Introduce Solution III (potassium acetate) to neutralize the pH; precipitating genomic DNA and proteins while keeping plasmid DNA in the solution.
Centrifuge and Precipitation:
Centrifuge to separate insoluble components from plasmid DNA in the supernatant.
Precipitate plasmid DNA by adding ethanol, followed by further washing and resuspension in Tris buffer.
Key Chemical Components
Solution I: Isotonic buffering to re-suspend cells.
Solution II: Sodium dodecylsulfate (SDS) destroys cell membranes and denatures proteins; sodium hydroxide denatures DNA.
Solution III: Potassium acetate neutralizes the lysate, helping to form a precipitate of genomic DNA and proteins while keeping plasmids in solution.
Ethanol: First, 95% ethanol interacts with sodium ions to precipitate DNA from solution; 70% ethanol washes excess salts.
Extraction of High Molecular Weight DNA
The process for extracting DNA from bananas is less stringent compared to plasmid extraction. Following an initial homogenization of the fruit, the protocol involves filtering through a coffee filter and layering ethanol to isolate DNA.
Protocol Steps
Homogenization: Mash banana in a baggie with water, salt, and soap to break down cell walls.
Filtration: Filter the mixture through coffee filter to separate DNA from cellular debris.
Precipitation: Add cold ethanol to the filtrate to extract DNA.
Scoop out DNA: Using a pipette, collect visible DNA precipitate and store appropriately.
Safety and Cleanup
When conducting these experiments:
Follow aseptic techniques to avoid contamination and misuse of equipment. Wear appropriate personal protective equipment (PPE).
Ensure proper disposal protocols are adhered to for biological waste, maintaining lab safety standards.
Conclusion and Connections
This lab integrates techniques crucial in molecular biology, applying knowledge of DNA structure and properties. Skills acquired here have wider implications in genetic research and applications, showcasing the intersection between theoretical knowledge and practical, hands-on experience in biological research.
This laboratory focuses on extracting plasmid DNA from E. coli and high molecular weight (HMW) DNA from fruits, specifically bananas. The following objectives guide the activities:
Extraction Techniques: Learn how to extract DNA using its special properties. This helps you understand the science behind DNA extraction.
Mini Plasmid Prep: Purify plasmid DNA so it can be used in future labs, especially for transforming competent cells, which means getting new DNA into cells for experiments.
Protocol Design: Use what you've learned to create a plan for extracting DNA from bananas, knowing how plant cells work and how to get DNA from them more easily.
Equipment Utilization: Get hands-on experience with common lab tools like micropipettors, microcentrifuges, and aseptic techniques, which help keep your samples clean and free of contamination.
Background & Importance
DNA is very important because it holds the genetic information that keeps cells functioning properly. Knowing how to extract DNA is key in molecular biology, helping with things like genetic engineering and studying antibiotic resistance. This lab allows you to work with live cultures, giving you a practical understanding of methods used in changing genes and doing molecular biology research. Learning these processes also builds your problem-solving skills and critical thinking abilities in a lab setting, which are vital for future science studies.
Types of DNA
Prokaryotic DNA
Bacterial Genomic DNA: Contains the genes necessary for survival, about 4000 of them, and is shaped like a circle attached to the cell membrane, allowing for efficient copying and use of genes.
Bacterial Plasmid DNA: Smaller, circular DNA that can carry about 2-25 genes. While these plasmids are not essential for survival, they can help bacteria gain special abilities, such as resistance to antibiotics, making them useful in biotechnology.
Eukaryotic DNA
Found in a straight line within the nucleus of cells. For humans, there are 23 pairs of chromosomes, totaling about 3.2 billion base pairs. Extracting DNA from eukaryotic cells is usually more complicated because of how tightly packed the DNA is and how it interacts with proteins.
Understanding Plasmid Extraction Steps
Overall Procedure
The plasmid extraction involves several steps:
Culture Preparation: Move E. coli from liquid culture into small tubes and spin them to collect the cells at the bottom.
Resuspension: Use STE (sterile TE buffer) to rinse and mix the bacterial cells, ensuring they prepare well for the next step.
Lysis of Cells: Add Solution I (an isotonic solution) to help balance the concentration in the cells.
Add Solution II (a mix of detergent and sodium hydroxide) to break open the cells and release their contents, including DNA.
Neutralization: Use Solution III (potassium acetate) to balance the acid/base levels, which helps separate out the genomic DNA and proteins while keeping plasmid DNA in the liquid.
Centrifuge and Precipitation: Spin the mixture to separate solid ingredients from plasmid DNA in the liquid above.
To get the plasmid DNA, add ethanol, wash away any impurities, and dissolve it in Tris buffer to prepare for future use.
Key Chemical Components
Solution I: Helps re-suspend cells in a balanced solution.
Solution II: Detergent (SDS) breaks open cells, and sodium hydroxide unravels DNA to release it.
Solution III: Potassium acetate balances the mix, helping to recover plasmids while eliminating larger genomic DNA and proteins.
Ethanol: Precipitates DNA from solution by removing water and salts, first using 95% ethanol and then washing with 70% ethanol to enhance purity.
Extraction of High Molecular Weight DNA
The process for extracting DNA from bananas is more straightforward compared to plasmid extraction. After mashing the fruit, the procedure involves filtering and using ethanol to isolate the DNA.
Protocol Steps
Homogenization: Mash the banana in a bag with water, salt, and soap to break open cells and release the DNA.
Filtration: Filter out the mash using a coffee filter to keep the DNA and remove unwanted bits.
Precipitation: Add cold ethanol to the filtered liquid to make the DNA visible and separate it from the solution.
Scoop out DNA: Use a pipette to collect the DNA you can see and store it properly.
Safety and Cleanup
When doing these experiments:
Always use proper methods to keep everything clean and avoid mixing in bacteria. Wear the right protective equipment like gloves and goggles.
Make sure to follow safety rules for disposing of biological waste to keep the lab safe and clean.
Conclusion and Connections
This lab combines important techniques used in molecular biology, applying what you know about DNA structure and how it works in real situations. The skills you learn in this lab are valuable for genetic research and practical applications, making it easier to connect theoretical knowledge with hands-on experience in science. Understanding these methods will help you in future studies and careers in biological research.