L12. DNA Extraction

On Monday 26th of January we did a new experiment about DNA extaction. The objective of this experiment was to study the DNA structure and to understand the process of extracting DNA from a tissue.

MATERIALS

- 600 ml Beaker
- 10 ml graduated cylinder
- Small funnel
- Glass stirring rod
- 10 mL pipet
-Safety goggles
-Cheesecloth
-Automatic pipet
-Kiwi
-Pineapple juice
-Distilled water
-90 % ethanol (ice cold)
- 8 ml DNA buffer (25 ml dish soap, 7 g NaCl, 450 ml tap water)

PROCEDURE

First of all we prepared the buffer in a 600 ml beaker. We put 450 mL of tap water, 25 ml of dish soap and 7g of NaCl and stirred the mixture careful because we don't want any foam or bubbles to form! 

1-We peeled the kiwi and chopped it to small pieces. We placed them in one 600 ml beaker and smashed them with the pestle to become a juice puree. 
2- We added 8 ml of the buffer to the mortar.
3- We mashed the kiwi puree carefully for 1 minute without creating many bubbles.
4- Then we filtered the mixture: we put the funnel on top of the graduated cylinder. We placed the cheesecloth on top of the funnel and added all the contents of the mortar carefully on top of the cheesecloth to fill the graduated cylinder. The juice drained through the cheesecloth but the chunks of kiwi didn't pass through.
6- We added the pineapple juice to the green juice (4 ml because we had 20 ml of green mixture DNA solution). This helped to obtain a purer solution of DNA because pineapple juice contains an enzyme that breaksdown proteins.
7- Next, we tilted the graduated cylinder and poured in an equal amout of ice-cold ethanol with an automatic pipet. We put the ethanol through the sides of the graduated cylinder to form a clear layer on top of the DNA solution.
8- We placed the graduated cylinder to eye level and using the stirring rod we stirred only the ethanol and DNA came up. 

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

QUESTIONS
1- What did the DNA look like?
 The DNA looks like long, small, white and thin fibers.  

2-Why do you mash the cells? Where it is located inside the cells?
Because you want to libreate the DNA that is located inside the nucleus.

3- Explain what is the function of every compound in the buffer
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 has to do with the method of extraction?  
This means that we can only see the DNA in the part of the ethanol because if it touches the water it will dissolve.

On Monday 2nd of February we repeated this experiment but we extracted the DNA from our own cells from our mouths. We did the exact same procedure but instead of mashing the kiwi we took mineral water with salt and we rinsed our mouth with it. At the end we observed the DNA in a microscope. 

L.11 Cytochrome C Comparison Lab

PROTEINS AND EVOLUTION

Genes are made of DNA and are inherited from parent to offspring. Some DNA sequences code for mRNA which, in turn, codes for the amino acid sequence of proteins. 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. The cytochrome C is a small protein found loosely associated with the inner membrane of the mitochondrion. It is found in eucariotic cells and has an hemeprotein. It is essential to the electrone transport chain and it is involved in using energy in the cell (ATP).

The purpose of this practice is to compare the relatedness between organisms by examining the amino acid sequence in the protein.

METHOD

First, we compared the amino acid sequence of Cytochrome C in various organisms:
-horse, donkey, whale (mammals)
-penguin, chicken (birds)
-snake (reptile)
-moth (insect)
-wheat (plant)
-yeast (fungi)

1- We marked the amino acids which were different.
2-Then we counted and recorded the total number of differences.
3- We shared the data with the rest of the class to complete Table 1.


After that, we made a cladogram (branching tree)
1- The two most closely related species had the fewest differences in amino acid sequence. We placed the two most closely related species on the two shortest branches of the tree.
2-Then we placed the next two closest species on the next shortest branches.
3- And we continued until all the species had been placed.

RESULTS AND OBSERVATIONS

CONCLUSIONS

There are 0 differences between chickens and turkeys in their Cytochrome C amino acid sequence.

We think that the horse and the zebra have 1 or 2 differences, like the donkey and zebra.
To make this prediction we used this information: if they can reproduce and if the offspring will be fertile or not, we compared organs, compared embrios...

More closely related organisms have more similar Cytochrome C because evolutionarily, it hasn't been that long since they separated. If the species are close, it means that less time has passed since they separated (there are less accumulated mutations). They have a common ancestor.

Other data, including genes, suggests that fungi are more closely related to animals than plants. But Cytochrome C data suggests that fungi, plants and animals are equally distantly related because if there are more than 40 genes, there are too many mutations to see it clearly.