1. Isolation of DNA
Biotechnology

2. Introductory Question
Do all organisms have the same amount of DNA? Explain.

3. Essential Question
How is DNA extracted from eukaryotic cells?

4. Purpose of Isolation Procedure
Purify DNA – have no other macromolecules –lipids, carbohydrates, proteins, RNA present

5. Studying DNA
Must first break open cells of the biological organism to release chromosomes (where DNA housed)
DNA is isolated (precipitated) from the solution and dissolved

6. Fruit DNA Isolating DNA from different fruit Why fruit?
Fruit are often polyploid meaning that they contain more than two copies of each chromosome
A chromosome is where the DNA is housed
By being polyploid, they have lots more DNA than an organism that has only two copies of each chromosome
For example, strawberries are actually octoploid meaning they have 8 copies of each chromosome rather than diploid (two copies of each chromosome) like most organisms

7. Dissolving Cell Membrane
Addition of detergent
Dissolves cell membranes composed of phospholipids
Denatures (unfolds) many proteins

8. NaCl-sodium chloride
Addition of NaCl is to raise the salt concentration
Under high salt, helps the cellular debris and proteins to precipitate (become a solid and fall out of solution)

9. Cell Bursts Dissolving cell membrane bursts open cells
Releases cellular contents into test tube
Precipitated proteins drop to the bottom of the vessel
Addition of heat speeds up denaturation process

10. Protease
Protease found in meat tenderizer –degrade protein contaminants in sample
Protease act as scissors cutting up proteins into smaller pieces

11. Centrifugation
Separate the precipitated proteins and cellular debris from the DNA that is still in solution
During centrifugation, the solid cellular debris and protein are pulled to the bottom of the tube
After centrifugation, the precipitated proteins and cellular debris will be the pellet (solid) at the bottom
DNA will be in the supernatant- liquid solution on top

12. Centrifugation

13. Ethanol The DNA in the supernatant is not visible
Must add ethanol to precipitate DNA (cause the DNA to come out of solution)
The DNA will look like stringy white fibers
Can spool on a glass rod

14. Dissolve Precipitated DNA
DNA dissolved in 10 mM Tris, 1 mM EDTA Buffer pH 8
Also known as TE buffer pH 8
Purpose of EDTA
Forms complexes with several metal ions (ex. Mg 2+)
Metal ions required for majority of DNases
Contaminating DNases will cut up isolated DNA
Without Metal ions, DNases will not work-cannot cut up DNA
Protects DNA from DNase degradation

15. DNA Isolation Lab
Each member of lab group will choose a different fruit
Perform the DNA isolation protocol
Spool DNA and weigh
Dissolve in TE buffer
Exchange data between members of the group