DNA EXTRACTION

Introduction

-Deoxyribonucleic 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 nucleobase (G, T, C, A) as well as a monosaccharide and a phospate group.
-These nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next.


Materials

- 1L Erlenmeyer flask
- 100mL beaker
- 10mL graduated cylinder
- Small funnel
- Glass stirring rod
- 10mL Pipet
- Knife
- Safety goggles
- Cheesecloth

- Kiwi
- Banana
- Pineapple juice (1mL / 5mL)
- Distilled Water
- 90% Ethanol ice-cold
- 7mL DNA buffer
  50mL dish soap
  15g NaCl
  900mL tap water


Objectives

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

Procedure

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

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

1. Peel the kiwi/banana 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 beaker. 

3. Later mash the kiwi/banana puree carefully for 1 minute without creating many bubbles. 

4. Finally 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 chessecloth but the chucks of kiwi/banana will not pass through into the graduated cylinder. 

6. Add the pineapple juice to the green juice (1mL of pineaplle juice to 5mL of DNA solution). This step will help us to obtain a purer solution of DNA. 

7. Tilt the graduated cylinder and pour in a equal amount of ethanol witg 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. 

(Dania's blog photo)

8. Place the graduated cylinder so that it is eye level. Using the stirring rod, collect DNA at the boundary of ethanol and kiwi/banana 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. 

RESULTS

QUESTIONS
1. Like a gelatine, white
2. To brake the cells, it's located in the cell wall
3. We add salt to brake the cells and soap to clean the proteins. Papaine cleans and delete the proteins.
4. It's a good method to catch it because the DNA goes to ethanol.

DETERMINACIÓ DE LA COMPOSICIÓ DE LA LLET (CASEÏNA, ALTRES PROTEÏNES, MIDÓ I GLÚCIDS REDUCTORS, PRESÈNCIA DE LÍPIDS)

Determinació de la llet

La llet conté vitamines, minerals, proteïnes, glúcids i lípids.
En aquesta pràctica identificarem tots aquests components i per tant serà un resum de totes les pràctiques d'identificació que hem realitzat al llarg del curs.


1.Determinació de la caseïna
La caseïna és una proteïna conjugada del tipus fosfoproteïna que se separa de la llet per acidificació i forma una massa blanca. Les fosfoproteïnes són un grup de proteïnes que es troben químicament unides a àcid fosfòric. La caseïna representa entre 77 i el 82% de les proteïnes presents a la llet. Aquesta proteïna presenta una baixa solubilitat (pH 4,6). En la primera part del experiment aïllareu la caseïna.

1- Afegim 200ml de llet en un vas de precipitats i l'escalfem fins 40 graus aproximadament.
2- El treiem del foc i afegim gota a gota àcid acètic (1ml d'àcid acètic glacial en 10 ml d'aigua destil.lada) amb un comptagotes. Agitem amb una vareta de vidre fins que acabi de precipitar tota la caseïna. (No afegir massa àcid acètic)

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3- Separem la caseïna amb l'ajuda d'una espàtula i la posem en un vidre de rellotge. I posem-lo assecar en la placa calenta.
4- Afegim immediatament en el líquid que ens ha quedat 4 gr de carbonat de calci en pols. L'agitem al llarg d'uns minuts i el guardem per la següent part. Això és el sèrum de la llet.
5- Quan la caseïna hagi perdut l'aigua calculem el percentatge de caseïna aïllada sabent que la densitat de la llet és 1,03 g/ml.



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2-Determinació d'altres proteïnes

Determinem la presència d'altres proteïnes en l'extracte (sèrum) de la pràctica anterior mitjançant la prova de Biuret. 

- 2ml de sèrum 

- 2ml d'hidròxid de sodi

- 5 gotes de sulfat de coure 

3- Determinació de midó i glúcids reductors

1.Determinem si la llet conté midó en el producte que hem extret (sèrum) de la pràctica anterior. 

2.Determinar la presència de glúcids reductors. 

- Petita quantitat de sèrum + lugol 

- Sudan III 

4- Determinació de la presència de lípids:

1.Determinem la presència de lípids de la llet en un tub d'assaig amb 2ml de llet. 

2.Al final afegim 1ml d'HCL al 50% al tub d'assaig anterior i l'escalfem suaument, finalment anotem els resultats que observem.

3.Provem-lo també amb el sèrum de la llet de la pràctica 1. 

- 2ml fehling A/B 

TAULA AMB ELS RESULTATS

PROTEIN DENATURATION

Introduction:

Denaturation is a process in wich 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 or compounds such as a strong acid or base, a concentrated inorganic salt or organic solvent.

Material:

2x250mL beaker

4 test tubes

Test tube rack

10mL Pipet

Knife

Glass marking pen

Potato

Distilled water

Hydrogen Peroxide

NaCl

HCL

Objectives: 

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

Procedure:

Catalase is a common enzyme found in nearly all-living organisms exposed to oxygen. It catalyzes the decomposition of hydrogen perioxide (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.

In this experiment we are going to test the catalse activity in different environment situations.  We are going to measure the rate of enzyme activity under various conditions, such as different pH values and temperatures. We will measure catalse activity by observing the oxygen gas bubbles when H2O2 is destroyed. If lots of bubbles are producted, it means the reaction is happening quickly and the catalase enzyme is very active. 

1. Prepare 30 ml  of H2O2 10% in a beaker (use a pipet) --> 10g H2O2     90g H2O

2. Prepare 30 ml of HCL 10% in a beaker. --> 10g HCl     90g H2O                       

3. Prepare 30 ml of NaCL 50% in a beaker. --> 50g NaCl  50g H2O

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. Inmerse 10 minutes your piece of potato inside HCl beaker

7. Inmerse 10 minutes another piece ofpotato inside NaCl 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:

Observations:

 

SAPONIFICATION

Introduction:
We used: NaOH, olive oil and Sudan III. 
With this material/elements we try to create soap.


Maerial
-Test tube rack
-Test tube
-Beaker
-Sterring rot
-Bunsen burner
-NaOH
-Olive oil
-Sudan III
-Water


Objective

1. To do the saponification
2. To make soap

Procedure:
Fisrt of all we take a test tube rack, test tube, sterring rot,... 
Later we take one test tube and put 2ml of NaOH and 2ml of olive oil. With the stirring rot, mix the solution. When you finish, put the test tube in a beaker with water and put it in the bunsen burner 10 minutes more or less. And you will see the soap (yellow).

Reaction of Saponifcation:

SECOND PART

Introduction:

We used milk and Sudan III to see if the colour change to red. 

Material:

-Test tube rack

-Test tube

-Beaker

-Dropper

-Milk

-Sudan III

Objective: 

1. Know if there are presence of lipids 

Procedure:

In the test tube we put milk but not much. Later we put some drops of Sudan III and we observe the results.

Observations: 

When whe put the Sudan III the colour of the milk change to red. That means that there are lipids in the substance. 

LIPIDS PROPERTIES

Introduction:
Lipids are heterogenous group of compounds synthesized by organisms that are present in all biological tissues. 
These compounds are characterized as natural substances that do not mix with water but dissolve in organic solvents. 
There are several classes of lipids, including: 

• Fatty acids
• Waxes
• Triacylglycerols
• Phospholipids
• Trepens ans steroids. 

Material:
Test tub rack
250mL beaker
Water
6 test tubes
Dropper
Scissors
Glass rod
Olive oil
Soap (detergent)
Ethanol 
Sudan III


Objectives: 

1. Test the solubility of lipids. 
2. Identify lipids in liquids compounds. 
3. Understand what are an emulsion and the effect of detergents 

Procedure: 
Solubility of some lipids:

Clean and dry three test tubes. Label as W (water), ethanol (E) and PE (ether)
Add 3 drops of oleic acid to 3 small test tubes. 
Add 1 ml of water in the first test tube (W)
Add 1 ml of ethanol in the second test tube (E). 
Add 1 ml of petroleum ether in the third test tube (PE). 
Shake carefully each test tube and record solubility and observations in your worksheet. 

Lipids identification:
Translucent mark:

Cut two pieces (10x10) of cellulose paper. 
Put 1 drop of water in the first squared piece. You will see a translucent spot, wait for a while and observe what is happening. 
Put 1 drop of olive oil in the second squared piece of cellulose paper. You will see a translucent spot. Wait for a while and observe what is happening.

Sudan III dye: BE CAREFUL SUDAN RED CAN STAIN CLOTHES!!!!

Sudan III is a red fat-soluble dye that is utilitzed in the identification of the presence of lipids, triglycerides and lipoproteins in liquids. 

Take the W test tube of the first experiment and add 2 drops of Sudan III 
Prepare four test tubes: 3 with milk with different fat content (M1, M2, M3) and soda (S). Add two drops of Sudan III.




PERMANENT EMULSION: 

Take a 250ml beaker and put 100ml of water. 
Add 1ml of olive oil. With a glass rod stir the mixture vigorously and let it stand for a few minutes. 
Make note of what is happening. 
Add 2 drops of soap and stir the mixture again. Let it stand for a few minutes and notice the differences between both mixtures.