L11. DNA EXTRACTION

1- Introduction 

DNA is a nuclic acid that encodes the genetic instructions used in the development and fuctioning of all living organsims and many viruses.
Nucleic acids are biopolymers formed by simple units called nucleotids. Each nucleotide is composed by a nitrogen-containing nucleobase (G, T, C, A) as well as a monosaccharide (deoxyribose) and a phosphate group.
Most DNA molecules consist of two strands coiled around each other to form a double helix. The two strands run in opposite directions to each other and are therefore anti-parallel. Moreover the bases of the two opposite strands unit according to base pairing rules : A-T and G-C.

2- Material

- 1L Erlenmeyer flask.

- 100mL beaker.

- 10mL graduated cylinder.

- Small funnel.

- Glass stirring rod.

- 10mL pipet.

- Knife.

- Safety goggles.

- Cheesecloth.

- Kiwi.

- Pineapple juice (1mL/5mL).

- Distilled water.

- 90% Ethanol ice-cold.

- 7mL DNA buffer.

- 50mL dish soap.

- 15g NaCl.

- 900mL tap water.

3- Procedure 

Put the ethanol in freezer you will need it really cold later. 

Prepare the buffer in a 0,5L beaker: add 450 mL of a tap water, 25 mL of dish soap and 7g NaCl. 

1- Pell the kiwi and chop it to small pieces. Place the pieces of the kiwi in one 600mL beaker and smash with a fork until it becomes a juice puree.

2- Add 8mL of buffer to the mortar.

3- Mash the kiwi puree carefully for 1 minutewithout creating many bubbles.

4- Filter the mixture: put the funnel on top of the graduated cylinder. Place the cheesecloth on top of the funnel. 

5- Add beaker contain carefully on top of the cheesecloth to fill the graduated cylinder. The juice will drain through the cheesecloth but the chucks of kiwi will not pass through into the graduated cylinder.

6- Add the pineaple juice to the green juice (you will need about 1mL of pineaple juice to 5mL of the green mixture DNA solution). This step will help us to obtain a purer solution DNA. Pineaple juice contains an enzyme that breaks down proteins.

7- Tilt the graduated cylinder and pour in an equal amount of ethanol with an automatic pipet. Put the ethanol through the sides of the graduated cylinder very carefully.You will need about equal volumes of DNA solution to ethanol.

8- Place the graduated cylinder so that it is eye level. Using the stirring rod, collect DNA at the boundary of ethanol and kiwi juice. Do not stir the kiwi juice; only stir in the above ethanol layer!!

9-  The DNA Precipitate looks like long, white and thin fibers.

10- Gently remove the stirring rod and examine what DNA looks like.

4- Questions

1- What did the DNA looks like? 

The DNA looks like long, small white and thin fibers.
 

2- Why do you mash the kiwi? Where it is located inside the cells?
Because we  wanted to liberate the DNA that is located inside the nucleus.
 

3- Explain what is the function of every compund of the buffer (soap and salt).
 The salt breaks the nucleus and the cell and the soap takes away the proteins.

4- DNA is soluble in water, but not in ethanol. What does this fact have to do with our method of extraction?  

This means that we can only see the DNA in the part of the ethanol beucause if it touches the water it will dissolve.

L10. PROTEINS AND EVOLUTION

1- Introduction

Genes are made of DNA and are inherited from parent to offspring. 
Soma DNA sequences code form RNA which turns codes for the amino acid sequence of proteins. Cytochrome C is a protein involved in using energy in the cell. Cytochrome C is found in most, if not all, known eukaryotes. Over time, random mutations in the DNA sequence occur. As a result, the amino acid sequence of Cytochrome C also changes. Cells without usable Cytochrome C are unlikely to survive. 

Cytochrome C is associated with the inter membrane of the mitochondrion. It is a small protein from eucaryote cell.  

2-Procedure 

We compare the protein "Cytochrome C": 

3-Conclusions

-Mamales: horse, whale and donkey

-Birds: chicken and penguin

-Reptils: snake

-Insect: moth

-Fungi: yeast

-Plant: wheat

	Horse	Donkey	Whale	Chiken	Penguin	Snake	Moth	Yeast	Wheat
Horse	0
Donkey	0	0
Whale	5	4	0
Chiken	11	10	9	0
Penguin	13	12	10	3	0
Snake	21	29	18	18	19	0
Moth	24	23	22	23	23	26	0
Yeast	40	39	39	40	39	40	46	0
Wheat	38	37	36	39	39	37	40	43	0

(taken from laura's blog)

(taken from myriam's blog)

L9. PROTEIN IDENTIFICATION

1-Introduction:

Biuret's test is a chemical test used for detecting the presence of peptide bonds. ( covalent bond formed between two peptides when the carboxyl group of the first one reacts with amino group of the second peptide). Polypeptides as proteins, are chains of amino acids link together by peptides bonds. 

A peptide bond can be broken by hydrolysis (the adding of water); and in organims, the protein molecules called enzymes makee the process easier.

The biuret reaction can be used to assess the concentration of proteins. The intensity of the colour is directly proportional to the protein concentration. 

The solution to be tested is treated with a strong base followed by a few drops of copper (II) sulphate. 

-If the solution turns purple, it will mean that the protein is present. But only peptides with a chain of at least two peptide bonds give a significant measurable colour shift with these reagents.

Proteins in the alkaline environment reduce Cu2+ to Cu+, which forms a coordination complex with proteins, leading to a blue to pink-purple colour change.

2-Objectives

- Identify peptides bonds.

- Compare protein concentration in different foods.

3-Materials:

- 7x250mL

- 6 test tubes

- test tube rack

- 6x10mL pipet

- mortar

- glass marking pen

- gloves

- goggles

- milk

- rice milk

- egg

- yogurt

- potato

- distilled water

- NaOH 20%

- 10 drops of CuSO4

4-Procedure: 

We are going to determine protein concentration in: milk, rice, yogurt, potato and egg. You've to be careful, cause we are going to test separately the egg white and the yolk. 

First of all we are going to dilute the protein.

1. Add 100mL of distilled water to each 250mL beaker. Label them with (M, RM, EW, EY, Y, P).

2. Separate the egg white and the yolk in another beaker.

3. Smash the potato in a mortar and add some amount of the smashed potato to the P beaker. 

Prepare the samples: 

1. Add 10mL of a dispersion of each food (M, RM, EW, EY, Y, P) to the indicate beaker. Calculate the final concentration. All the groups will use the same dispersion from the beakers.

2. Prepare 6 test tubes and label them. Add 2mL of each test tube of the every food dilution of each beaker.

3. Add 2mL of 20% NaOH dissolution.

4. Shake gently and add 5 drops of CuSO4 in each tube. Allow the mixture to stand for 5 minutes.

5. Note any colour change. Remember that proteins will turn solution pink or purple.

6. Compare the test tubes. 

5-Results and conclusions:

-Milk: positive
-Rice milk: negative
-Egg white: positive
-Egg yolk: negative
-Yogurt: positive
-Potato: positive ------- (it's not correct because potato had starch so it had to be negative.) 
The positive result, are the foods of animals, because all they have proteins. Rice milk have starch like potato so they are negative. 

We ordered from more concentration to less concentration according to the colour. 
1. Egg white.
2. Potato and yogurt.
3. Milk.

6-Questions:

1.- Which food has protein?
Egg white, milk, yogurt, potato, (the animal food)

2.- Which food has more protein? why?
1 Egg white

2 Yogurt 

3 Cow milk 

3.- Do you find any difference between rice and cow milk?
 Cow milk has animal proteins and rice milk has also protein but vegetal ones. 

4.- Is there any difference among milk and yogurt? why?
Yes, yogurt has more protein than milk. Because yougurt has ben fermentade.