DNA Isolation Flashcards
Name the bond found between paired bases of the DNA double helix
Hydrogen bond
What is DNA soluble in?
Water
What is DNA insoluble in?
ethanol
What is the purpose of using NaCl in the lab?
NaCl helps to mask the negative charge of the DNA molecule, allowing the DNA to clump together
What is the purpose of using detergent in the lab?
To break down the phospholipid bilayer in the cell membranes and release the contents of the cell
What material is used to breakdown the phospholipid bilayer?
Detergent
What are monomers of DNA called?
Nucleotides
What inhibits the breakdown of DNA during precipitation?
Cold temperatures
What is an enzyme that degrades DNA?
DNase
List the materials used to isolate DNA
120 mL soft fruit (strawberries)
resealable sandwich bag/ziploc bag
10 mL liquid dish detergent
60 mL ice-cold 99% isopropanol
3.75 mL NaCl
120 mL warm water
What organism was the DNA isolated from? give latin and common names
strawberries
Fragaria ananassa
Why were the strawberries broken down by hand first?
To mechanically break down the rigid plant cell walls
What was the purpose of adding the NaCl and warm water solution to the strawberries?
To dissociate the DNA from the histone proteins
What are 3 reasons why the strawberry/NaCl/water mixture was placed in hot water for 15 minutes?
To denature the proteins
To dissolve the lipids in the cells
To inhibit the enzymes
What was the purpose of putting the strawberry/NaCl/water mixture into ice-cold water for 10 minutes?
To precipitate the proteins and lipids out
Why was the solution filtered through a cheese cloth?
To filter out the proteins, lipids, and cell walls
What occurred when the isopropanol was poured slowly over the fruit filtrate? What is the purpose of this?
A clear layer of isopropanol formed on top of the fruit filtrate
DNA will diffuse into the alcohol/precipitate out because alcohol is less polar than water
What happened when the fruit filtrate was mixed with the isopropanol?
The DNA precipitated onto the stir stick and the isolated DNA became visible
Briefly explain the procedure of this experiment
- put 120 mL of fruit in plastic bag and remove all air
- macerate fruit in plastic bag with hands (2 mins)
- prepare 3% NaCl solution by dissolving 3/4 tsp of NaCl in 120 mL of warm water
- add 60 mL of NaCl solution to fruit bag
- Add 10 mL of liquid detergent to fruit solution
- squeeze solution for 1 minute
- Heat 1 L of water to ~60 degrees C
- place sealed fruit bag into hot water, cover for 15 mins
- put fruit bag into bowl of ice cubes and water for 5-10 mins
- strain fruit solution through cheese cloth into clean cup
- transfer fruit filtrate into clear cup
- tilt cup and pour ice cold isopropanol into cup to form layer
- gently mix at the interface using a stir stick
What % NaCl was the solution with warm water?
3% NaCl
How does a cell package ~6 feet of DNA into the nucleus?
The structure of DNA allows it to be packaged tightly
It is a double helix that is coiled into small beads by histone proteins
Is the white sticky precipitate you isolated pure DNA? What else could be present? Why?
No, it is not pure DNA because there may still be proteins BUT the two big contaminants are: RNA and sodium
RNA will be present because it is isolated by the same method as DNA
Positively charged Na is still attracted to the negatively charged DNA
Define DNA
It is the macromolecule that carries genetic information required for the growth, development, function and reproduction of living organisms
Describe the structure of DNA
DNA is made up of nucleotides that pair to form a double-stranded molecule
What does each eukaryotic chromosome consist of?
a single linear double-stranded DNA molecule wrapped around proteins called histones
Describe a chromatin
the DNA-protein complex consisting of a single linear double-stranded DNA molecule and histone proteins
What do chromosomes look life for most of the cell cycle?
A diffuse mass of long thing chromatin fibres
When a cell prepares to divide (mitosis or meiosis), what happens to the chromatin?
It condenses to form distinct chromosomes
T or F: DNA is present in the cells of only some organisms
False. DNA is present in the cells of ALL organisms
Many food plants are ___ploid
Polyploid
What does polyploid mean?
the organism has more than two copies of their genome in each cell
What makes food plants ideal for DNA extraction?
Each cell contains a lot of DNA because food plants are polyploids = they have more than 2 copies of their genome in each cell
What ploidy are strawberries?
octoploid (8n)
Why is it better to use a soft fruit like strawberry or kiwi for this experiment?
It is easier to release the DNA from the nucleus without breaking it
