DNA EXTRACTION
Detergent
eNzymes
Alcohol
Purpose of
DNA
extraction…
Summarize this
purpose in your
lab book.
Isolation of DNA is often the first
step before:
• DNA profiling
• cloning
• disease diagnosis
• DNA sequencing
• genetically modified organisms (GMO) agriculture, pharmaceutical
• Environmental testing, bioterrorism
… so it is an
important lab
skill to learn.
Laboratory
Protocol
Create a table like this in which you fill in the steps of
the procedure and the biological explanation behind
what is occurring at each step:
Protocol Directions
1.
2.
Explanation
Laboratory
Protocol
1. Obtain the cup containing 10 ml Gatorade from
your instructor.
2. Take the Gatorade from the cup into your mouth,
and swish the water around vigorously for 60
seconds. Gently chew the insides of your
cheeks. It is NOT helpful to drawblood!
3. Carefully expel the liquid into the plastic vial.
4. Dispose of the paper cup in the trash.
Cell
Collection
Gently chewing the
inside of the mouth
combined with a water
mouth wash is used to
dislodge epithelial cells
lining the mouth
Ample cell collection is
critical for success.
Why
Gatorade?
•
Gatorade contains
salt
• Na+ ions of NaCI
bind to the
phosphate groups
of DNA molecules,
neutralizing the
electric charge of
the DNA
molecules.
• The addition of
NaCI allows the
DNA molecules to
come together
instead of
repelling each
other.
O
Na+
O
Na+
P
O
Base
O
O
CH2
Sugar
O
Na+
Na+
O
P
O
Base
O
O
CH2
Sugar
OH
Laboratory
Protocol
5. Obtain the detergent from your workstation, and
add 2 ml of detergent to your plastic vial.
6. Place the cap on the vial and gently invert your
tube 5 times (don’t shake it!).
7. Let your vial sit
undisturbed for 10 minutes.
Why Detergent?
To break open the cell and nuclear membranes, allowing
the DNA to be released into the solution
Laboratory
Protocol
8. Add a pinch of meat tenderizer to your vial.
9. Place the cap on your vial
and gently invert it a few
times.
10. Let sit for 10 minutes.
• Meat tenderizer is made of enzymes that
break down proteins in meat.
Why Add
Meat
Tenderizer?
• In this case, the enzymes destroy
proteins that bind DNA.
• This treatment increases the amount of
intact DNA that is extracted.
Laboratory
Protocol
11. Gently pour COLD ethanol into your vial so that
it forms a layer on top of the Gatorade solution
12. Let sit for 10 minutes.
Adding Ice
Cold
Alcohol?
• DNA does not dissolve in alcohol.
• The addition of cold alcohol makes the DNA
clump together and precipitate out of
solution.
• Precipitated DNA molecules appear as long
pieces of fluffy, stringy, web-like strands.
• Microscopic oxygen bubbles “aggregate” ,
or “fuse” together, as the DNA precipitates.
• The larger, visible air bubbles “lift” the DNA
out of solution.
Post Lab
Questions
ANSWER IN COMPLETE SENTENCES
1. DNA is a microscopic molecule. How
come we can see it?
2. List five potential applications of DNA
extraction.