L11. DNA extraction

Introduction:

Deoxyribonucleic acid (DNA)  is a nuclic acid that encodes the genetic instructions used in the development and fuctioning of all known living organsims and many viruses.
Nucleic acids are biopolymers formed by simple units called nucleotids. Each nucleotide is composed of 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.

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.

 

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. Stir the mixture. 

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.

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 you want to liberate the DNA that is located inside the nucleus.
 

3- Explain what is the function of every compund of the buffer (soap ans 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

Introduction: 

Genes are made of DNA and are inherited from parent to offspring. Soma DNA sequences code form RNA which, in turn, 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.  

Procedure and conclusions: 

 

We compare the protein "Cytochrome C": 

 

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

(from laura's blog)

(from myriam's blog)

 

L9. Protein denaturation

Introduction:

Denaturation is a process in which proteins or nucleic acids lose the quaternary, tertiary amd secondary structure that is present in their native state. Is the result of the application of some external stress or compounds such as strong acid or base, a concentraced inorganic salt or organic solvent.

If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death.  Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to communal aggregation. 

In very few cases denaturation is reversible and proteins can recuperate their native state when the denaturing factor is removed, this process is calledrrenaturation.

Catalase is a common enzyme found in nearly all-living organisms exposed to oxygen. It catalyzes the descomposition of hydrogen peroxide (H2O2) to water and oxygen. It is a very important enzyme in protecting the cell from oxidative damage and preventing the accumulation of hydrogen peroxide.

   2 H₂O_{2 --------------------->}   2 H₂O + O<sub>2

Is a tetramer of four polypeptide chains, ecah over 500 amino acids long. It contains four Porphyrin Heme groups (iron groups) that allow the enzyme yo react with the hydrogen peroxide. The optimum pH for human catalase is aprox. 7, in other organisms vary between 4 and 11. The organelle that stores catalase in eukaryotic cells is the peroxisome, which also contains peroxidases.</sub>

 Material:
 
- 2x250mL beaker.
- 4 test tubes.
- Test tube rack.
- 10 mL pipet.
- Knife.
- Glass marking pen.
- Potato.
- Distilled water.
- Hydrogen Peroxide.
- NaCl.
- HCl.

Procedure:

We are going to test the catalase activity in different enviroment situations. we are measures the rate of enzyme activity under varios conditions, such as different pH values and temperature. We will measure catalase activity by observing the oxygen gas bubbles when H₂O₂  is destroyed. If lots of bubbles are produced, it means the reaction is happening quickly and the catalaseenzyme is very active. 

1. Prepare 30mL of H₂O₂  10% in a beaker (use a pipet).
2. Prepare 30mL of HCl 10% in a beaker.
3. Prepare 30mL of NaCl 50% in a beaker.
4. Peel a fresh potato tuber and cut the tissue in five cubes of  1cm³. Weigh them and equal the mass.
5. Label 5 test tubes (1,2,3,4,5).
6. Immerse 10 minutes your piece of potato inside HCl beaker.
7. Immerse 10 minutes another piece of potato NaOH beaker.
8. Boil another piece of potato.
9. With a mortar, mash up the third piece of potato.
10. Prepare 5 test tubes as indicated below:

     

    1.- Raw potato

    2.- Boiled potato

    3.- Potato with HCl 

    4.- Potato with NaCl

    5.- Mashed up potato
     
11.  Add 5mL H₂O₂  10% in each test tube.
12.  With a glass-marking pen mark the height of the height of the bubbles.

13. Parts:	In this experiment this was...
    Independent variable	Tratament of each potato.
    Dependent variable	The height of the bubbles.
    Experimental Group(s)	Boiled, with HCl, with NaCl and mashed up potato.
    Control Groups	Raw potato.
    Constants	Weight, same amount of H2O2 , time...

    (From Myriam's blog)
    
    
    
    
      
     
     
     
     
     
     
     
     
     Questions: 
    1- How did the temperture of the potato affect the activity of catalase? 
    2- How did the change of the pH of the potato affect the activity of catalase? 
    3- In which potato treatment was catalase the most active? Why do you think this was?