Protocol for DNA Extraction

Learning objectives

1. Understand that DNA is the material in the cell’s nucleus that contains instructions for making proteins.

2. Understand that numbers of chromosomes and amount of DNA can vary widely among different species, in part due to chromosome doubling (polyploidy) events.

3. Understand that DNA isolation requires breaking down membranes to release DNA.

4. Demonstrate ability to perform hypothesis testing. Be able to:

   1. State a hypothesis for how a process is expected to work.

   2. State a prediction for what should occur if hypothesis is correct.

   3. Compare an observation to a prediction and decide whether hypothesis is supported or refuted

Experimental goal

Predict whether rice or wheat germ has more DNA based on background information regarding the species’ genome size. Test the predictions by extracting DNA from both and qualitatively comparing the outcome.

Step 1: Put a bottle of 91% isopropyl acohol in the freezer or a cooler with ice the night before you do the expermiment.

It needs time to get cold (it will not hurt it to stay in the freezer for days or weeks).

Background

DNA is the storage molecule for hereditary information in plants and all other living organisms. DNA includes the instructions to make proteins, and the proteins made based on these instructions accomplish nearly all important tasks in cells. A gene is one section of DNA that usually contains the instructions to make one protein. In plants, animals and all other eukaryotes (complex organisms whose cells have membrane-bound organelles) most DNA is stored in the cell’s nucleus, although mitochondria and chloroplasts also contain much smaller amounts of DNA. The dominant life stage of vascular plants is diploid, meaning that for every kind of chromosome, there are two copies in the nucleus. The entirety of all of the DNA in an organism is called its genome.

All plants need to accomplish similar tasks and have similar protein needs. Nonetheless, they vary widely in the size of their genome. This variation occurs for a couple of reasons. First, only a small fraction of DNA is made of genes that code for proteins. Some of the remaining DNA helps with the process of allowing the genes to be used to make proteins, but much of it does nothing at all. However, miuch of the DNA does nothing at all, and different amounts of this non-functional DNA accumulates in different species.

A second reason that DNA content varies so widely is polyploidy. Polyploidy refers to when rare problems during cell division that result in increasing of the number of chromosomes. One common form of polyploidy is a doubling of chromosome number, which results in the dominant generation of a polyploid plant having 4 copies of each chromosome rather than two copies (called a tetraploid; see the figure). While those 4 copies are initially very similar to one another, they eventually evolve some differences and begin operating as 2 pairs of chromosomes rather than one set of four. Each time polyploidy occurs, it doubles the size of the genome. Over long enough time periods, polyploidy can happen repeatedly, each time again doubling the size of the genome.

Genome size can be measured by counting the number of DNA nucleotide base pairs across all of the chromosomes in the genome Genomes are large – the human genome is larger than 3,000 megabases, and a megabase (Mb) is equal to 1,000,000 base pairs. That means the human genome is larger than 3,000 x 1,000,000 base pairs, equaling more than 3 billion base pairs! Plant genomes vary widely in size and can be larger or smaller than this.

Rice and wheat are both grasses and important food crops. We refer to their seeds as grains. Grains have three parts:

1. starchy endosperm tissue, which is the largest portion of the seed as serves as a storehouse of nutrition. It is made of large cells.

2. a protective seed coat around the outside made of large fully developed cells.

3. an embryo, which is the immature stage that develops into the adult plant. The cells in the embryo divide quickly and never become large. Although the cells are small, they have exactly the same genome size (the same amount of DNA) as the large adult cells they will eventually become.

[add labeled image of monocot seed here]

While rice and wheat are both grasses, their genomes differ. Rice is a diploid, whereas wheat is a polyploid. Specifically, wheat is a hexaploid, which means that it has 6 of each type of chromosome (the prefix hexa means 6).

Hypothesis / Prediction

Create a hypothesis about whether wheat germ or rice germ has more DNA for the same quantity of each material. Your hypothesis should be an explanation, in the following form: “I hypothesize that ________________ will have more DNA than a similar quantity of ___________ because___________(explain fully)__________________________________________ “.

Now, create a specific prediction for this experiment. The experiment we will perform involves extracting DNA from similar amounts of wheat and rice germ and comparing the amount of DNA that results from each. Your prediction should be in the following form: “If my hypothesis is correct that… (fill in your hypothesis here), then I predict that I will detect greater amounts of DNA in the extract from ___________ than from ____________.”

Save these – you will record your hypothesis and prediction as part of the DNA extraction assignment on Blackboard.

Pre-lab questions

Next complete the pre-lab questions on Blackboard. After you have completed them with a 70% or higher score, you will be granted permissions to view and complete the DNA extraction protocol.

[Students complete 8-question quiz on this background information on Blackboard. When they have completed this with score of > 70%, they are allowed to progress to second Blackboard "test" where they enter their hypothesis and predictions (not timed, graded by hand afterwards). When they have completed their entries for this, they are then able to see instructions for the lab protocol.

Experiment Protocol

Supplies provided to you:

1. (1 g) wheat germ and (1 g) rice germin Ziploc-style sandwich bags

2. 4 plastic Ziploc-style bag (1 qt)

3. gentle laundry detergent (similar to Woolite)

4. 2 coffee filters

5. 2 disposable flat bottom 50 mL tubes

6. 2 bamboo skewers

7. Gloves

Supplies you provide

1. 91% isopropyl alcohol (chilled in feezer or on ice – see below)

2. freezer or cooler/bowl of ice to chill alcohol

3. scissors (you can use your scalpel if necessary)

Safety

• Wear goggles and gloves while handling detergent and isopropyl (rubbing) alcohol.

Instructions

• • 1. Place isopropyl alcohol in freezer. If freezer is unavailable, procure ice, put in cooler or bowl and chill at least 3 tablespoons (40 mL) of isopropyl in the ice. It is fine to chill the entire bottle of alcohol if that is easier. Allow to chill for an hour, as the protocol does not work with warm alcohol.

    2. You will perform the first steps of disrupting the rice and wheat germ samples inside their plastic bags.

    3. Use provided disposable tube to measure (20 mL = 1.5 tablespoons) warm water (warm enough to feel warm but touch comfortably), and add this amount to both bags. Also add half of the provided detergent to each bag (3 mL = ½ teaspoons per bag). Seal the bag. Detergents disrupt hydrophobic interactions between lipids, and we are using them to disrupt the cell membrane as well as nuclear, mitochondrial, and chloroplast membranes in order to release DNA.

    4. Being careful not to unseal bag, crush the rice and wheat germ contents thoroughly. Crush & mix both bags for a similar duration and amount of effort (2 – 3 minutes).

1. Make a funnel out of an empty plastic bag by cutting the bag diagonally corner to corner. Retain the bottom section of the bag. Cut the intact corner off the bag – this will be the small opening of the funnel (see photo).

   1. 1. Label the two disposable large (50 mL) test tubes, one as rice germ and one as wheat germ. Put a coffee filter into the funnel. Place the funnel over one of the test tubes (you can place the test tube in a glass to hold it upright).

1. Pour the contents of the appropriate bag into the filter and allow the liquid to filter through and into the test tube. The solids should remain in the filter. Repeat for the second tube. The DNA is dissolved in the liquid, so it passes through the filter into the test tube.

   1. 1. Photographs are shown, but using different plant materials.

1. The next step is to separate the DNA from the liquid. To accomplish this, angle the test tube and pour 1.5 tablespoons (15 mL) ice-cold 91% isopropyl alcohol slowly down the side of the tube. DNA will precipitate out of solution (leave its dissolved state) and float at the interface between the alcohol and the liquid. It is important to pour slowly down the side of the tube because if the alcohol mixes too much with the liquid, the DNA will not separate. (Photographs are shown using different plant materials.)

1. Use one bamboo skewer to gently stir the contents of each tube. You should see white viscous matter at the interface of the extraction and alcohol layers.

   1. Photograph the results of both tubes:

      1. Arrange the tubes next to each other with the rice germ on the left and wheat germ on the right. Label the tubes with the permanent marker provided in your lab supplies ("wheat" and "rice").

      2. Take the photograph level from the side of the tube with good light, and let the tubes fill as much of the frame as possible.

      3. Make sure the labels are showing and an identifying sticker is visible.

Results & Discussion - You will do this part

• Using your photo, make a qualitative comparison between the amount of DNA visible in each extraction (by qualitative, I mean judge visually; we cannot measure the results on a numeric scale). Which tube appears to have more DNA, and is the difference large or small? (The DNA is floating at the surface of the liquid in both test tubes). Save the photo above on your computer by right clicking on it and choose "save image as" and choosing a folder to save it in and give it a name you will remember.

On the Blackboard pre-lab quiz, you were asked to hypothesize whether the rice or wheat germ has more DNA based on the background information provided. Now compare your hypothesis to the actual genome size of rice and wheat, shown in this figure. Explain whether the hypothesis you previously made matches the data in this linked graph.

• Assess whether the outcome was consistent with your prediction. If your prediction was supported, remember that your hypothesis still is not proven because science does not prove hypotheses correct – instead it only can support or refute them. Consider what you could do to further test your hypothesis. If your prediction did not match your results, then suggest a new hypothesis that is a proposed explanation for what occurred.

• Finally, consider a closely related question that you could ask in a similar experiment. Your experiment could test a change in methods that you think might make the protocol work better, or it could test a question related to the amount of DNA in different substances. In a paragraph, describe what you would test, what are your hypotheses and predictions and how you would test it.

What to turn in - Complete #1 below

1. Create a word processing document (.doc or .docx format): Include the following images & responses:

   1. Your initial hypothesis & prediction

   2. Labeled photographs of the results of the wheat-germ and rice germ extractions. (use the one provided with smiley face)

   3. A description of the results of both tubes.

   4. A statement of whether your results concurred with your prediction.

   5. Depending on your results, describe in a paragraph one of the following:

      1. If your results did not match your prediction, then suggest why you think there was a discrepancy. Explain either how you would modify your hypothesis or how you would modify the experiment if you think a problem with the experiment led to the results not matching your prediction.

      2. If your results did match your prediction, suggest an interesting additional hypothesis that a future student in this course could test related to this question. It cannot be exactly what we already did, and it should be something achievable in a home setting. This completes the research cycle, as results almost always lead to new questions.

2. Upload labeled photographs of both experimental results to the Padlet page. That way, we can compare results with classmates. (do not do the Padlet portion, #2).

References:

The idea of preparing the initial steps of the extraction in Ziploc-style bags was from Imagination Station (http://imaginationstationtoledo.org/educator/activities/extract-dna)

Final reflection questions (turned in as assignment on Blackboard)

Qualitative Comparison of Amount of DNA in Rice Germ and Wheat Germ: Photos & Answers

Paste in the specified photos and answer the questions below.

1. What was your initial hypothesis & prediction?

2. Paste in a labeled photograph of your wheat germ and rice germ extraction (use the photo that was provided with the smiley face in the DNA extraction part II document).

3. Describe and compare the results for the two extractions. Was there a clear difference in the amount of DNA and if so, in which direction?

4. Depending on your results, describe in a paragraph one of the following:

a. If your results did not match your prediction, then suggest why you think there was a discrepancy. Explain either how you would modify your hypothesis or how you would modify the experiment if you think a problem with the experiment led to the results not matching your prediction.

b. If your results did match your prediction, suggest an interesting additional hypothesis that a future student in this course could test related to this question. It cannot be exactly what we already did, and it should be something achievable in a home setting. This completes the research cycle, as results almost always lead to new questions.