P12. DNA EXTRACTION

INTRODUCTION:

• Desoxyribonucleic acid (DNA)  is a nucleic acid that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses.

• Nucleic acids are biopolymers formed by simple units called nucleotides. Each nucleotide is composed of a nitrogen-containing nuclease (G, T, C, A) as well as a monosaccharide (desoxyribose) and a phosphate group.

• These nucleotides are joined to one another in a chain by covalent bonds between the sugar of the nucleotide and the phosphate of the next.

• 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.

•  Within cells, DNA is organized into structures called chromosomes.

OBJECTIVES:

1. Study DNA structure
2. Understand the process of extracting DNA from a tissue.

MATERIALS:- 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 the freezer, you will need it really cold later!Prepare the buffer in 0,5L beaker: Add 450mL of tap water, 25mL of dish soap and 7g NaCl. Stir the mixture.

1. Peel 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 pure.2. Add 8mL of buffer to the mortar. 3. Mash the kiwi puree carefully for 1 minute without 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 cheesecloth to fill the graduated cylinder. The juice will drain through the cheesecloth but the chucks of kiwi will not pass through in to the graduated cylinder.6. Add the pineapple juice to the green juice ( you will need about 1mL of pineapple juice to 5mL of the green mixture DNA solution). This step will help us to obtain a purer solution of DNA . Pineapple juice contains an enzyme that breaks down the 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 the ethanol and kiwi juice; only the 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 the DNA looks like. 

QUESTIONS:1.- What did the DNA looks like?

• The DNA looks like white  and thin fibers.

2.- Why do you mash the kiwi? Where it's located inside the cells?

• The DNA is located in the nucleus and we mashed it to liberate it. 

3.- Explain what is the function of every compound of the buffer (soap and salt). 

• The salt can breaks the nucleus cell and we put soap to take 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?

• We can see the DNA in the part of ethanol because, if we touch the water the DNA can dissolve.

P11. 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. 

Objectives:

1. To compare the relatedness between organisms by examining the amino acid sequence in the protein, Cytochrome C.

Conclusions and coments:

QUESTIONS:

  1.- How many Cytochrome C amino acid sequence differences are there between chickens and turkeys?

•        0 differences.

         2.- Make a branching tree for chikens, penguins, and turkeys. 

         

•          Penguins--Turkey----3 differences.
•          Turkey-- Chicken----0 differences

3. a. Predict the number of Cytochrome C amino acid sequence differences you would expect to see between 

• - horse --- zebra: 1 or 2
• - donkey --- zebra: 1 or 2

b. what other information did you use to make this prediction? 

• They can reproduce, the offspring is fertile, comparing the organs or anatomic proofs, comparing embryos.

5. List three other things used to determine how organisms are related to each other:

• If they can reproduce, the offspring is fertile, comparing the organs or anatomic proofs, comparing embryos.

6. Explain why more closely related organism have more similar Cytochrome C.

• Not so long ago that there have been separated and many mutations. There are fewer differences in DNA.

7. Other data including other genes, suggests that fungi are more closely related to animals than plants. What are some reasons that the Cytochrome C data suggests that fungi, plants, and animals are equally distantly related?

• If you have more than 40, suffered a lot of mutations and can not be compared. If Compares other proteins, there are other changes and we can not draw more conclusions.

P10. Protein Denaturation

Introduction:

• Denaturation is a process in which proteins or nucleic acids lose the quaternary, tertiary and secondary structure that is present in their native state. Denaturation is the result of the application of some external stress (heat and pH change) or compounds such as a strong acid or base, aconcentrated 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. This last effect results from the bonding of the hydrophobic proteins to reduce the total area exposed to water.  
• In very few cases denaturation is reversible and proteins can recuperate their native state when the denaturing factor is removed.
•  This process is called renaturation.

Objectives:

1. Study the relation between the structure and the function of proteins.
2. Understand how temperature, pH, and salinity effect to the protein structure.

Hypothesis:

• Catalase denaturated proteins.

Materials:

• 2x250 mL beaker
• 4 test tubes
• Test tube rack
• 10 mL pipet
• Knife
• Glass marking pen
• Potato
• Distilled water
• Hydrogen Peroxide
• NaCl
• HCl

Procedure:

Prepare 30mL of H₂O₂  10% in a beaker (use a pipet), put 30mL of HCl 10% in a beaker.

Prepare 30mL of NaCl 50% in a beaker.

Peel a fresh potato tuber and cut the tissue in five cubes of  1cm³. Weigh them and equal the mass.

Label 5 test tubes (1,2,3,4,5).

Immerse 10 minutes your piece of potato inside HCl and NaOH  beaker.

Boil another piece of potato.

Mash up the third piece of potato.

Prepare 5 test tubes: one of raw potato, boiled potato, potato with HCl, potatp with NaCl, and mashed up potato.

Add 5mL H₂O₂  10% in each test tube.

With a glass-marking pen mark the height of the height of the bubbles.

Compare the results of the test 5 test tubes.

Conclusions and coments:

The temperature and acidity are more able reducing the salinity.

• Independent variable: treatment of each potato (pH, temp....)
• Dependent variable: the height of the bubble
• Experimental Group: The rest test tubes
• Control Groups: Raw

RESULTS:

• Mashed: 74mm
• NaCl: 39mm
• HCl: 35mm
• Boiled: 37mm
• Raw: 40mm

P9. Protein identification

Introduction:


We will identify proteins and look at what foods carry more. So use the Biuret reagent.

Objectives:

1. Identify peptide bonds
2. Compare protein concentration in different foods

Hypothesis:

• The egg white proteins have more than yogurt, which have more protein than milk, which have more protein than potatoes. Rice milk and yolk have not proteins.

Materials:

• 7x250 mL beaker
• 6 test tubes
• Test tube rack
• 6x10 mL pipet
• Mortar
• Glass marking pen
• Gloves
• Goggles
• Milk
• Soy Milk
• Egg
• Yogurt
• Potato
• Distilled water
• NaOH 20%
• 10 drops of CuSO4

Procedure:

Take 10 mL of normal milk (M), rice milk (RM), egg white (EW), yolk (EY), yogurt (Y) and potato (P)., And put in a test tube (each food in a different tube).
Put 2 mL of 20% NaOH.
Let CuCO4 5 drops of each tube.
Hopefully 5 minutes.
Comparing the tubes: the more purple, more protein.

Conclusions and coments:

These are the results of practice. (proteins are sorted from most to least):

EY: -
EW: 1
M: 3
RM: -
P: 4
Y: 2

Questions:

1- Which food has proteins?
Eggwhite, milk yogurt and potato, animal food.

2- Which food has more proteins? Why?
Eggwhite because it's animal food and it's rich in proteins. 

3-Do you find any difference between rice milk and cow milk?
Rice milk doesn't have proteins however cow milk does as i had said before.

4- Is there any difference among milk and yogurt? why?
Yes, yogurt has more protein than milk because the protein is more concentrated than in the milk, it has less water.