1. DNA ISOLATION: Strawberry Lab

2. DNA Food Facts Each cell contains 9 feet of DNA
In an average meal, you EAT approximately 55,000,000 cells
This is equal to approximately 93,205 miles of DNA

3. DNA
DNA (deoxyribonucleic acid) is found in the nucleus of all eukaryotic cells
Most cells have a DIPLOID (2n, 2 copies of each chromosome) chromosome number
Many plants are POLYPLOID (contain several sets of chromosomes)
Strawberries are octaploid (8n)

4. DNA cont.
DNA is enclosed in a nuclear and a cell membrane made of phospholipids
DNA is also coiled around proteins called HISTONES
Both the phospholipid layer and the proteins must be removed in order to access the DNA

5. Isolation Background…
DNA is present in the cells of all living organisms.
The process of isolating DNA from a cell is the first step for many laboratory procedures in biotechnology (PCR, sequencing, etc.)
The scientist must be able to separate DNA from the unwanted substances of the cell gently enough so that the DNA does not DENATURE (break up)

6. Isolation Background cont.
Most DNA extraction protocols consist of two parts:
A technique to lyse the cells gently and solubilize the DNA
Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules

7. A comparison of DNA extraction methods used in research labs as versus in classroom labs
Lysis: grind in Liquid N2 and use detergent
Precipitation Part I: phenol/chloroform extraction to get rid of proteins
Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA
Precipitation Part III: addition of ethanol to pull DNA out of solution
Wash and Resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.
Classroom
Lysis: grind by hand and use detergent & salt
Filtration: Use of filter paper to remove larger cell parts
Precipitation: addition of ethanol to pull DNA out of solution
Wash and Resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.

8. LYSIS
In DNA extraction from plants, this step commonly refers to the breaking of the cell wall and cellular membranes (most importantly, the plasma and nuclear membranes)
The CELL WALL (made of cellulose) is disrupted by mechanical force (ex: mashing)
Then the addition of a detergent in the which breaks down the cell membranes
DETERGENTS are able to disrupt membranes due to the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules. The detergent molecules are able to pull apart the membranes
The end result of LYSIS is that the contents of the plant cells are distributed in solution

9. Lysis cont. Cell membranes are made of phospholipids
Phospholipids won’t dissolve in water
What do you use in your home to remove oils from your hands or your dishes?
SOAP or DETERGENT!

10. Lysis cont.
Soap gathers between oil and water capturing the oil in a bubble called a MICELLE
When you wash your hands, the soap carries the oils away

11. Lysis cont.
By adding a small amount of table salt (NaCl) to the soap solution, the solution can punch holes in the nuclear and cell membranes
The soapy solution also helps removes proteins

12. Lysis cont. DNA is tightly coiled around proteins called histones
Proteins must be removed to see DNA
Enzymes, like the ones found in meat tenderizer, can remove protein

13. Filtration
The contents of the cell (organelles, proteins, etc.) must be separated from the DNA
The larger cell parts can be removed by filtering the solid from the liquid

14. PRECIPITATION
This a series of steps where DNA is separated from the rest of the cellular components
The addition of salts during lysis which interrupt the hydrogen bonds between water and DNA molecules, allows it to precipitate better
The DNA is precipitated from the protein with isopropanol or ethanol
In the presence of cations (Na+ from the salt), ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.
The DNA can pelleted by spinning with a centrifuge and the supernatant removed
Note: because this protocol does not use phenol/chloroform, the DNA extracted in a classroom lab is not as “clean” as the DNA extracted in a research lab!

15. Precipitation cont.
To see DNA, it must be extracted or “spooled” from the remaining liquid you filtered
DNA dissolves in water, but NOT in alcohol
Adding COLD ALCOHOL will cause DNA to PRECIPITATE (separate out) from the liquid filtrate

16. Precipitation cont.
The DNA will appear as a white precipitate once the alcohol is added
HOLD THE TUBE by the TOP, not the bottom so the DNA strands won’t fragment from the heat of your hands!

17. Precipitation cont. DNA is sticky and will adhere to other surfaces
A glass stirring rod can be used to spool (remove) the DNA by using a turning motion

18. Washing and Resuspension
The precipitated DNA is laden with acetate salts. It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but not resuspend the DNA.
Resuspension:
The clean DNA is now resuspended in a BUFFER (provide appropriate pH and ions) to ensure stability and long term storage. The most commonly used buffer for resuspension is called TE.