Category: Curriculum Map

 

Introduction:

Since DNA is the blueprint for life, everything living contains DNA. DNA isolation is one of the most basic and essential techniques in the study of DNA. The extraction of DNA from cells and its purification are of primary importance to the field of biotechnology and forensics. Extraction and purification of DNA are the first steps in the analysis and manipulation of DNA that allow scientists to detect genetic disorders, produce DNA fingerprints of individuals, and even create genetically engineered organisms that can produce beneficial products such as insulin, antibiotics, and hormones.

DNA can be extracted from many types of cells. The first step is to lyse or break open the cell. This can be done by grinding a piece of tissue in a blender. After the cells have broken open, a salt solution such as NaCl and a detergent solution containing the compound SDS (sodiumdodecyl sulfate) is added. These solutions break down and emulsify the fat & proteins that make up a cell membrane. Finally, ethanol is added because DNA is soluble in water. The alcohol causes DNA to precipitate, or settle out of the solution, leaving behind all the cellular components that aren’t soluble in alcohol. The DNA can be spooled (wound) on a stirring rod and pulled from the solution at this point.

Objective:

To extract DNA from cells.

Materials:

Blender, split peas, salt, detergent, water, measuring cup and spoons, strainer, meat tenderizer, alcohol, test tube, glass stirring rod

Procedure:

1. First, you need to find something that contains DNA such as split peas, fresh spinach, chicken liver, onion, or broccoli.

2. Measure about 100 ml or 1/2 cup of split peas and place them in a blender.
3. Add a large pinch of salt (less than 1 ml or about 1/8 teaspoon) to the blender.
4. Add about twice as much cold water as the DNA source (about 200 ml or 1 cup) to the peas in the blender.
5. Blend on high (lid on) for about 15 seconds.

6. The blender separates the pea cells from each other, so you now have a really thin pea-cell soup.

And now, those 3 easy steps:

7. Pour your thin pea-cell soup through a strainer into another container like a measuring cup or beaker.

8. Estimate how much pea soup  you have and add about 1/6 of that amount of liquid detergent (about 30ml or 2 tablespoons). Swirl to mix.

8. Let the mixture sit for 5-10 minutes.

The detergent captures the proteins & lipids of the cell membrane.

9. Pour the mixture into test tubes or other small glass containers, each about 1/3 full.
10. Add a pinch of enzymes to each test tube and stir gently. Be careful! If you stir too hard, you’ll break up the DNA, making it harder to see. (Use meat tenderizer for enzymes. If you can’t find tenderizer, try using pineapple juice or contact lens cleaning solution.)

The DNA in the nucleus of the cell is molded, folded, and protected by proteins. The meat tenderizer cuts the proteins away from the DNA.

11. Tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea mixture. Pour until you have about the same amount of alcohol in the tube as pea mixture.

12. Alcohol is less dense than water, so it floats on top forming two separate layers.
13. All of the grease and the protein that we broke up in the first two steps move to the bottom, watery layer.
14. DNA will rise into the alcohol layer from the pea layer. You can use a glass stirring rod or a wooden stick to draw the DNA into the alcohol.
15. Slowly turning the stirring rod will spool (wrap) the DNA around the rod so it can be removed from the liquid.

Questions:

1. Does the DNA have any color?

2. Describe the appearance of the DNA.

3. Do only living things contain DNA? Explain.

Frequently Asked Questions: 1. I’m pretty sure I’m not seeing DNA. What did I do wrong?

First, check one more time for DNA. Look very closely at the alcohol layer for tiny bubbles. Often, clumps of DNA are loosely attached to the bubbles.

If you are sure you don’t see DNA, then the next step is to make sure that you started with enough DNA in the first place. Many food sources of DNA, such as grapes, also contain a lot of water. If the blended cell soup is too watery, there won’t be enough DNA to see. To fix this, go back to the first step and add less water. The cell soup should be opaque, meaning that you can’t see through it. Another possible reason for not seeing any DNA is not allowing enough time for each step to complete. Make sure to stir in the detergent for at least five minutes. If the cell and nuclear membranes are still intact, the DNA will be stuck in the bottom layer. Often, if you let the test tube of pea mixture and alcohol sit for 30-60 minutes, DNA will precipitate into the alcohol layer.

2. Why does the DNA clump together?

Single molecules of DNA are long and stringy. Each cell of your body contains six feet of DNA, but it’s only one-millionth of an inch wide. To fit all of this DNA into your cells, it needs to be packed efficiently. To solve this problem, DNA twists tightly and clumps together inside cells. Even when you extract DNA from cells, it still clumps together, though not as much as it would inside the cell.

Imagine this: the human body contains about 100 trillion cells, each of which contains six feet of DNA. If you do the math, you’ll find that our bodies contain more than a billion miles of DNA!

3. Can I use this DNA as a sample for gel electrophoresis?

Yes, but all you will see is a smear. The DNA you have extracted is genomic, meaning that you have the entire collection of DNA from each cell. Unless you cut the DNA with restriction enzymes, it is too long and stringy to move through the pores of the gel; instead, all you will end up seeing is a smear.

4. Isn’t the white, stringy stuff actually a mix of DNA and RNA?

That’s exactly right! The procedure for DNA extraction is really a procedure for nucleic acid extraction. However, much of the RNA is cut by ribonucleases (enzymes that cut RNA) that are released when the cells are broken open.

 

 

FERMENTATION – MAKING ROOT BEER David Fankhauser’s Main Page

 

Introduction:

Fermentation has been used by mankind for thousands of years for raising bread, fermenting wine and brewing beer. The products of the fermentation of sugar by baker’s yeast Saccharomyces cerevisiae (a fungus) are ethyl alcohol and carbon dioxide. Carbon dioxide causes bread to rise and gives effervescent drinks their bubbles. This action of yeast on sugar is used to ‘carbonate’ beverages, as in the addition of bubbles to champagne).

We will set up a fermentation in a closed system and capture the generated carbon dioxide to carbonate root beer. You may of course adjust the quantities of sugar and/or extract  (Zatarain’s) to taste. 

	EQUIPMENT	SUPPLIES
	clean 2 liter plastic soft drink bottle with cap funnel 1 cup measuring cup 1/4 tsp measuring spoon 1 Tbl measuring spoon	Cane (table) sugar [sucrose] (1 cup) Zatarain’s Root Beer Extract (1 tablespoon) (When I could not find it locally, I ordered a case of 12 bottles for $18 from Zatarain’s, New Orleans, LA 70114 powdered baker’s yeast (1/4 teaspoon)  (Yeast for brewing would certainly work at least as well as baking yeast.) cold fresh water

 

 

INSTRUCTIONS:

	1) Assemble the necessary equipment and supplies
	2) With a dry funnel, add in sequence: 1 level cup of table sugar (cane sugar) (You can adjust the amount to achieve the desired sweetness.)
	3) Add: 1/4 teaspoon powdered baker’s yeast ( fresh and active) (Fleischmann’s or other brand)
	4) You can see the yeast granules on top of the sugar.
	5) Shake to distribute the yeast grains into the sugar.
	6) Swirl the sugar/yeast mixture in the bottom to make it concave (to catch the extract).
	7) Add with funnel: 1 Tbl of root beer extract (I prefer Zatarain’s, but Hires, etc. will work.) on top of the dry sugar
	8) The extract sticks to the sugar which will help dissolve the extract in the next steps.

	9) Half fill the bottle with fresh cool tap water (the less chlorine, the better). Rinse in the extract which sticks to the tablespoon and funnel. Swirl to dissolve the ingredients.
	10) Q.s. [fill up] to the neck of the bottle with fresh cool tap water, leaving about an inch of head space, securely screw cap down to seal. Invert repeatedly to thoroughly dissolve.

If you leave it in a warm temperature longer than two weeks, you risk an explosion…

11) Place at room temperature about three to four days until the bottle feels hard to a forceful squeeze. Move to a cool place (below 65 F). refrigerate overnight to thoroughly chill before serving. Crack the lid of the thoroughly chilled root beer just a little to release the pressure slowly. NOTE: Do not leave the finished root beer in a warm place once the bottle feels hard. After a couple weeks or so at room temperature, especially in the summer when the temperature is high, enough pressure may build up to explode the bottle! There is no danger of this if the finished root beer is refrigerated.

12) Move to a refrigerator overnight before opening.

 

NOTE: There will be a sediment of yeast at the bottom of the bottle, so that the last bit of root beer will be turbid. Decant carefully if you wish to avoid this sediment.

A WORD ABOUT THE ALCOHOL IN HOME MADE ROOT BEER: The alcoholic content which results from the fermentation of this root beer and found it to be between 0.35 and 0.5 %. Comparing this to the 6% in many beers, it would require a person to drink about a gallon and a half of this root beer to be equivalent to one 12 ounce beer. I would call this amount of alcohol negligible, but for persons with metabolic problems who cannot metabolize alcohol properly, or religious prohibition against any alcohol,  consumption should be limited or avoided.