Biology DNA Lab lab answers PDF

Preview

Summary

Biology DNA Lab lab answers and all the steps. Report...

Description

1

DNA Extraction Lab

Adam Sharifi, Abhira Khindaj and Enes Dede

Ms. Cook

June 4, 2021

2

Purpose: The purpose of this lab is to extract DNA from several different sources for comparison purposes. Hypothesis: If the DNA from an equal amount of strawberry ,banana and an onion were extracted, then the banana sample would yield the most DNA and the onion sample would yield the least amount of DNA. Background Information: DNA is an essential molecule that is found in all living organisms. DNA stores genetic information that helps synthesize proteins, which are crucial for performing vital bodily functions. DNA is a complex macromolecule and scientists are still in the process of researching it. In order to study DNA, it needs to be extracted from the organism. DNA extraction is a process first done by scientist Friedrich Miescher in 1869 when he isolated DNA from a leucocyte (11). This was a scientific breakthrough as it allowed scientists to develop to efficiently extract DNA from living organisms (11). DNA extraction is important because it helps scientists to further understand the unique characteristics of the DNA molecule, such as structure, size and function (11). DNA extraction also plays an important role in biotechnology, as it allows scientists to be able to genetically engineer desired traits into plants or animals (12). Furthermore, DNA extraction is important in the research of genetic disorders such as cystic fibrosis and

3

Down syndrome (12), as well as research potential medication or treatment in order to cure the genetic disorder (11). Materials: ●

Small, resealable plastic bag

●

Measuring spoons/cups

●

Shot cup/old plastic test tubes

●

Washcloth

● Freezer ● Cups ● Toothpicks ● 70-95 % isopropyl ● Banana ● Strawberry ● Onion ● Clear detergent ● Table salt - Sodium Chloride Procedure: 1. Ensure the alcohol reagent is ice-cold by placing it in the freezer or sitting it in ice. 2. Use ¼ banana (and mash it using your fingers within a sealed plastic bag. 3. Measure 2 tablespoons (12mL) of 0.9% solution NaCl and mix thoroughly with bag contents. A 0.9% salt solution can be made by adding 1/4tsp (1.5g) of salt to

4

165mL of water and mixing until fully dissolved. This solution should be at room temperature. 4. Add 1 tablespoon (6mL) of dish-washing liquid (clear liquid if possible) and mix gently within the plastic bag to create minimal bubbles. Allow this mixture to sit for 5 min. 5. Strain the mixture from the plastic bag through cheesecloth (use several layers folded over each other) set into a funnel. Collect the liquid filtrate. If cheesecloth isn’t available, a clean washcloth could be used to separate the supernatant liquid from the solids. Squeeze the mass to collect the liquid in a clean cup. Dispose of the solids. 6. Transfer 1 teaspoon (2mL) of filtrate liquid to red a 15-20mL test tube or other tall cup such as a shot cup. If the container is long and thin, it will be easier to see when the DNA precipitates in the next few steps! 7. Add 1-½ tablespoons (8mL) of ice-cold 70-95% isopropyl alcohol by pouring it gently down the side of the tilted container. The alcohol layer should float on the filtrate. Do not mix but allow the solutions to layer. 8. Let the solution sit for 2-3 minutes. A white substance, DNA, will precipitate at the interface of the two liquids. 9. Carefully insert a toothpick and slowly swirl it to collect the DNA from the test tube. The DNA on the wooden skewer can be set on a piece of wax paper in in order to weigh it. Ensure that the wax paper is pre-weighed or that the scale is starred.

5

10. Repeat this procedure for strawberry, apple, or green pea samples of equivalent Volume. 11. Estimate the amount of DNA extracted from each sample type and do a quantitative comparison. This can be accomplished several ways. If a scale is available, the skewer or popsicle stick that is used to collect the DNA can be weighed before and after to determine the mass of DNA that is retrieved. Otherwise, a visual comparison can be made and the difference in volume can be estimated.

Pre-lab Questions: 1. Why is the banana treated with a soap solution? The soap solution helps break down the cell membrane of the cells, and will help release the DNA. The membranes are made up of lipids. Lipids are molecules that contain hydrocarbons, examples of which are oils and fats. The dish soap is amphiphilic, meaning it has a hydrophilic head and a hydrophobic tail. The amphiphilic properties of soap helps to break down the lipid bilayer and it opens up the cell membrane in order to extract the DNA. (2) 2. Why is alcohol carefully poured down the side of the test tube? By pouring the alcohol down the side of the test tube, it ensures that any CO2 is released before it enters the solution. We also do not want the alcohol to mix with the mixture so, the cold alcohol carefully poured down the edge of the glass causes the DNA to separate from the other biological components. (3) 3. What safety precautions must be taken when working with ethanol?

6

Ethanol is a colourless liquid that is highly flammable and has a slight odor. Consuming ethanol alone can lead to fatal health issues, for example, it can lead to a coma or even death. There are even some studies that suggest that ethanol may be carcinogenic meaning it can cause cancer. When working with ethanol, you should wear gloves, safety goggles, clothing covering your whole body and maybe even a face shield. (1) Ethanol has several safety risks, requiring the employment of several safety procedures while working with this material. To begin with, prolonged physical contact with ethanol may irritate both the skin and the eyes. Prolonged or recurrent skin contact with ethanol may result in skin dryness or cracking, as well as peeling, redness, and irritation (4). Also, ethanol is a flammable substance that can cause a fire. When conducting this experiment at home, the researcher should keep the ethanol as far away from any potentially flammable items as possible, such as an oven.

7

Proof: Figure 1. Strawberry DNA Extraction

Figure 2. Banana DNA Extraction

¼ of a banana undergoing lysis to the left and the DNA

8

extracted from ¼ of a banana to the right

Figure 3. Onion DNA Extraction

Some onion going through lysis

DNA extracted

9

Data Observations: Table 1: Characteristics of DNA extracted from fruit/vegetable samples Characteristics of

Banana

Strawberry

Onion

Transparent

White/Red Colour

White/transparent

extracted DNA samples Colour Quantity extracted

1.5

5-6

1

(using a green pea as reference) Structure

● Jelly type substance

It appears to be a chunk of gum.

Very sluggish and soft stringy

While in the shot ● Sticky ● Jelly type consistency

glass, it seems to

substance

have a circular/sphere form. Soft String substance.

Analysis Questions: 1. A number of steps are required to isolate DNA from cellular contents. Describe what happens at each step, and why it acts to separate the parts of the cell.

10

a. The first step to isolate DNA from cellular contents is to break the cell open to release DNA. This step is called Lysis. (8)The cells and nucleus are broken into to release the DNA.(9) This was done two different ways, one of which was where we smashed the fruit to release its contents. The second way was when we used the detergents to release more DNA from the nucleus. In the experiment, the dish soap added in step 4 acted as a detergent, breaking down the grease layer around the nucleus, releasing the DNA. The next step is to separate the DNA from the proteins and other debris.(9) This next step is called precipitation. (8) Precipitation is where the DNA is separated, the NA+ ions are going to neutralize the negative charges on the molecules of DNA. This is done so the DNA is more stable and not as water soluble.(8) In our experiment, this step is done when we add the 0.9% salt solution to the DNA mixture. This mixture is then strained with a cheesecloth or a tea filter. Then, the ice cold alcohol is added making the DNA to precipitate since it is not soluble in alcohol. After waiting for the DNA to settle, you are able to analyze the DNA. 2. Do you think the process or results would be different if you were to use a vegetable, or animal tissue. Explain! a. If DNA were taken from different organisms, it would appear the same. Because of the varied arrangement of the nucleotides, DNA varies from organism to organism. This order determines various genetic information, but it cannot modify the general structure of the DNA on a vast scale, as seen by the white precipitate formed in this lab.(6)

11

3. What property of DNA allows it to be ‘coiled’ around a glass rod once precipitated? a. The property of DNA that allows it to be “coiled” around a glass rod once it has precipitated is its supercoiled structure (13). When the DNA strands begin to precipitate, they will want to return to their original coiled state (13). Therefore, stirring the glass rod in a circular motion will cause the DNA to be coiled around the glass rod in order to return to their original coiled state (13). Furthermore, DNA is a polar molecule and so is the glass rod (13). The non-polar attraction between the DNA and the glass rod also allows the DNA to be coiled around the glass rod once precipitated (13). 4. Based on your procedure and results, how is DNA isolated from plant or animal cells? a. Based on the procedure, DNA is isolated from plant and animal cells by us mechanically breaking open the cells. We did this by mashing up the fruit, and then by using the dish soap, we were able to release the DNA from the nucleus and cell. Usually, the DNA could have dissolved in water, however, by adding the 0.9% solution of NaCl, we were able to neutralize the DNA. Then the cold isopropyl alcohol was used to layer on top of the mixture and the DNA is able to precipitate. Lastly we can use a toothpick or popsicle stick to gently remove the DNA out of the solution. (8) 5. Describe the appearance of the DNA that was collected.

12

a. The appearance of the strawberry DNA obtained was that it was reddish-whitish in colour, medium in size (not too little for the human eye and not too large), and the structure was a cylinder-like form after being detached from the liquid material. b. The appearance of the banana DNA obtained was that of transparent colour, small amounts of about 1.5 peas and the structure was like a jelly-like fluid. The DNA extracted was also sticky to the touch. c. The appearance of the onion DNA obtained was that of yellow-white colour, a small amount compared to one pea. It was stretchy, and sticky Extend Further: 1. What other steps could be taken in this procedure to better purify your DNA sample? a. Some other steps that could have been taken in this procedure to better purify the DNA sample is how we broke down the cell and nucleus. In the experiment we smashed the fruits in a plastic bag with our hands, something that we could have done instead was to put the fruit in the blender. This way the cell could be broken down fully to better release the DNA. Another step to better purify the DNA could have been to add meat tenderizers to the mixture at the same step we added the salt solution. The meat tenderizer helps keep the DNA structurally intact,control the enzymes and break down any proteins. (10) 2. How can you ensure that your sample of DNA does not contain any impurities, including other nucleic acids or proteins?

13

a. DNA must be separated from proteins and nucleic acids to ensure purity in DNA samples. Proteins can be stained by enzyme-containing detergents. Detergents act as a lysis solution containing proteinase K. Proteinase K can aid in the hydrolysis (decomposition of proteins and histones by interacting with water) of proteins and histones, allowing pure DNA to be left alone. (5) 3. How is polyploidism exploited by commercial agriculture, and is there a connection to this laboratory investigation? a. Polyploidy is when an organism has more than two complete sets of chromosomes (14). Polyploidism occurs when there is an error in mitosis or meiosis (like non-disjunction) that causes the formation of gametes that contain a complete set of duplicate chromosomes (14). Polyploidism is often exploited in commercial agriculture. One way that polyploidism is exploited is through mutation breeding (15). Mutation breeding is a breeding technique when farmers breed crops with the intention of creating a crop with a mutation that contains a desirable trait (15). Polyploidism helps with mutation breeding because of its increased number of genetic material, which increases mutation frequency during breeding (15). Furthermore, polyploidism is exploited to produce seedless plants (15). Seedless plants occur when the plant has an odd set of chromosomes (16). Therefore, farmers deliberately use breeding techniques to breed plants with odd sets of chromosomes in order for them to be seedless (16). There is a connection between polyploidism and

14

this lab, because fruits that are polyploid will contain more genetic material than those who are not, thus increasing the amount of DNA extracted. For example, a strawberry is an octoploid (contains 8 sets of chromosomes) and a banana is a triploid (contains 3 sets of chromosomes), therefore one would expect more DNA to be extracted from a strawberry than a banana (16). Source of Error: The possible sources of experimental error is the percentage of isopropyl alcohol used. For the strawberry and onion DNA extraction, 85% isopropyl alcohol was used whereas for the banana DNA extraction, 70% isopropyl alcohol was used due to that being the only source of isopropyl alcohol available. The difference in isopropyl alcohol concentration could have affected the possible amount of DNA precipitate formed. Furthermore, another potential source of experimental error is the amount of samples used. The strawberry and onion samples were being compared to ¼ of a banana. Due to human error, there could have been more or less of the strawberry or onion sample which would’ve affected the amount of DNA yielded from those samples.

Conclusion: Based on the findings of this lab, the hypothesis stated before the lab was conducted was incorrect. The hypothesis stated that the banana sample would yield the most amount of DNA however, it was the strawberry sample that yielded the most DNA. This experiment demonstrated that the strawberry sample yielded the most and the

15

onion sample yielded the least. This is because strawberries have large genomes, they are octoploid, which means they have eight of each type of chromosome in each cell. Thus, strawberries are an exceptional fruit to use in DNA extraction labs and strawberries yield more DNA than any other fruit (7). Whereas bananas are triploids (16) and onions are diploid (17), therefore they yield less amount of DNA due to having less amount of genetic material available. In further experiments, scientists can use this information to compare the DNA yield of plant cells and the DNA yield of animal cells. Furthermore, further experiments can be done to see the genetic similarities between the DNA samples extracted from fruit/vegetable samples.

16

Citations

1. Ethanol: Versatile, Common and Potentially Dangerous. (2014). MSDS Online. https://www.msdsonline.com/2014/04/21/ethanol-versatile-common-and-potential ly-dangerous/ 2. Melissa Wilson Sayres. (2016, April 19). Seeing DNA. ASU - Ask A Biologist. Retrieved May 27, 2021 from https://askabiologist.asu.edu/activities/banana-dna 3. Bailey R. How to Extract DNA From a Banana [Internet]. ThoughtCo. [cited 2021Mar20]. Available from: https://www.thoughtco.com/how-to-extract-dna-from-a-banana-373317#:~: text=The%20precipitation%20step%20(pouring%20the,by%20extraction% 20with%20the%20toothpicks 4. Hazardous Substance Fact Sheet [Internet]. 2016. Available from: https://www.nj.gov/health/eoh/rtkweb/documents/fs/0844.pdf 5. Buddies S. Squishy Science: Extract DNA from Smashed Strawberries [Internet]. Scientific American. Scientific American; 2013 [cited 2021Mar20]. Available from: https://www.scientificamerican.com/article/squishy-science-extract-dna-fro m-smashed-strawberries/ 6. Nature News. Nature Publishing Group; [cited 2021Mar19]. Available from: https://www.nature.com/scitable/topicpage/dna-is-a-structure-that-encodes-biol ogical-6493050/ 7. “DNA Extraction Lab: Strawberry.” NAU, www2.nau.edu/lrm22/lessons/dna_extraction/strawberry_dna.html#:%7E:text=S

17

trawberries%20have%20large%20genomes%3B%20they,%2C%20kiwi%2C%20etc. ).&text=DNA%20is%20not%20soluble%20in%20alcohol. Accessed 4 June 2021. 8. “The Basics of DNA Extraction – Alaska BioPREP Virtual Textbook.” University of Alaska, bioprep.community.uaf.edu/learning-modules/2-dna-extraction-4/the-basics-of-d na-extraction. Accessed 4 June 2021. 9. “DNA Extraction.” Science Learning Hub, www.sciencelearn.org.nz/resources/2036-dna-extraction. Accessed 4 June 2021. 10. “ DNA Extraction.” [ebook] Portland: OMSI. Available at: [Accessed 4 June 2021]. 11. “DNA Extraction Isolates DNA from Biological Material.” WhatisBiotechnology.org, www.whatisbiotechnology.org/index.php/science/summary/extraction/dna-extra ction-isolates-dna-from-biological-material#:~:text=The%20ability%20to%20extra ct%20DNA,environment%20and%20for%20determining%20paternity. 12. Erickson, Eric. “Common Uses for DNA Extraction.” Revolutionary Science, Revolutionary Science, 9 July 2020, www.revsci.com/blogs/biotechnology/common-uses-for-dna-extraction.

18

13. “DNA Extraction Lab.” Bartleby, Bartleby.com, www.bartleby.com/essay/DNA-extraction-lab-P3V9E29K6YYS#:~:text=So%20wh en%20the%20DNA%20is,coil%20around%20the%20stirring%20rod. 14. Woodhouse, Margaret, et al. “Polyploidy.” Nature News, Nature Publishing Group, 2009, www.nature.com/scitable/topicpage/polyploidy-1552814/. 15. Meru, Geoffrey. “5. Polyploidy.” PlantBreeding, 15 May 2013, plantbreeding.coe.uga.edu/index.php?title=5._Polyploidy#Mutation_breeding. 16. Bradish, Christine. “Polyploidy – or How Do We Get Seedless Fruit?” Sustainable, Secure Food Blog, 19 June 2020, sustainable-secure-food-blog.com/2019/05/07/polyploidy-or-how-do-we-get-see dless-fruit/#:~:text=Plants%20can%20have%20multiple%20sets%20of%20chrom osomes%2C%20which%20is%20called%20polyploidy.&text=Simple%20graphic% 20depiction%20of%20the,are%20diploid%2C%20and%20have%20seeds. 17. McLatchie, Jonathan. “Why the ‘Onion Test’ Fails as an Argument for ‘Junk DNA.’” Evolution News, Discovery Institute, 2 Nov. 2011, evolutionnews.org/2011/11/why_the_onion_test_fails_as_an/....