DNA EXTRACTION
Add table salt, large pinch (1 gram) or ¼
 is the isolation and purification of DNA
(deoxyribonucleic acid)
 Purpose: To obtain DNA in a relatively purified
form which can be used for further
investigations, i.e. PCR, sequencing, etc
DNA
 Deoxyribonucleic acid
 The complex chemical compound found in
chromosomes that contains the genetic code
Extracting DNA from any living thing
3 STEPS:
1. Detergent
 contains sodium laurel sulfate, which cleans
dishes by removing fats and proteins. It acts
the same way in the DNA extraction protocol,
pulling apart the lipids and proteins that
make up the membranes surrounding the cell
and nucleus. Once these membranes are
broken apart, the DNA is released from the
cell.
2. eNzymes
 to cut the Protein that surrounds the DNA to
free DNA
3. Alcohol
 Ethanol or isopropyl alcohol causes
the DNA to precipitate. When DNA comes out
of solution it tends to clump together, which
makes it visible.
Materials:
A. Extracting the DNA
1. Put alcohol in the freezer
2. Distilled water (250ml) pre heated to 60
degrees Celsius
3. Finely chop ½ cup of onion / strawberry /
kiwi put in a beaker and add the preheated
distilled water
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4.
teaspoon, cool then blend. (if no blender, put
in zip bag and mash vigorously)
5. Stir (delicately) until the mixture is of a thin,
soupy consistency
 For DNA to Escape one must break cell wall
 the role of the salt is to neutralize the charge of
the DNA's sugar phosphate backbone. This makes
the DNA less hydrophilic (less soluble in
water).
B. Strain the DNA mixture
1. Pour the thin cell mixture though the strainer
(cheesecloth / coffee filter paper) into
another large beaker/glass jar
2. Measure 12ml of the soup into a small
beaker/another glass jar
3. Add 2ml of liquid detergent
4. Swirl (daintily) to mix stir thoroughly using a
glass rod / clean barbecue stick
5. Let mixture sit in container with warm tap
water (60-65oC) for 5-10 mins
 Dishwashing detergent = lyse lipids
C. Add Meat Tenderizer Enzyme Powder
1. Add a pinch of enzyme (meat tenderizer) to
mixture. (using pineapple juice or contact
lens cleaning solution will do the same as
tenderizer)
2. Gently stir with toothpick / skewer for 5
mins, continue stirring, not too vigorously. Be
careful, if you stir too hard, you’ll break up
the DNA, making it harder to see
 Cut histones
 Meat tenderizer- acts as an enzyme to cut proteins
just like a pair of scissors because. The DNA in the
nucleus of the cell is molded, folded, and protected
by proteins.
D. Alcohol separation
1. Quarter-fill a large test tube/tall glass with
the mixture
2. Slowly pour the same amount of ice-cold (70-
95% isopropyl or ethyl alcohol) into the test
tube, pour the alcohol down the side of the
 test tube (tilt the test tube). It forms a layer  The salt that was added helps it stick
on top of the cell mixture. together. So what you see are clumps of
tangled DNA molecules
3. Do not mix the two layers together. Amount
 DNA normally stays dissolved in water, but
of alcohol and mixture should be the same, or
when salty DNA comes in contact with
the alcohol volume could be more than the
alcohol it becomes undissolved. This is
amount of the mixture
called precipitation.
4. Observe the mixture for a few mins. You will
Salt
see a white, threadlike substance rise from
the mixture to rest above the alcohol layer.  Sodium is a positive ion and often associates
This is the DNA that you have extracted. with negative ions as part of useful compounds.
 Salt help precipitate protein and carbohydrates
5. You can get more DNA to precipitate from the
away from the DNA
solution using DNA collecting tool (glass or
 Salt helps strip away the proteins associated
paper clip hook or cut inoculation needle)
with DNA. + and – charge
6. Gently lift the water solution up into the  Salt and detergents are used to break down cell
alcohol layer (this allows more DNA to get in walls and nuclear membranes to release the
contact with the alcohol and precipitate) DNA
 They work by chemically poking holes in the
7. If you want to save the DNA extracted, cell membrane or walls
transfer to a container filled with alcohol,  Once holes are poked in the membranes, the
leave glass container uncapped until the membranes can be further disrupted
ethanol has evaporated. Cover, refrigerate mechanically with a blender
with label.
Use of the detergent
 Cold ethanol helps the DNA to precipitate more
quickly.  Each cell is surrounded by a sack (cell
membrane)
RESULT:
 DNA is found inside the second sack (nucleus)
 DNA precipitates as a white stringy / snotty within the cell
film at the water- alcohol interface and
eventually will rise into the alcohol layer from  To see the DNA, we have to break it open
the mixture layer.
 A cell membrane has 2 layers of lipid
 Allow the test tube to sit for several minutes.
molecules with protein going through them.
The clearer the DNA, the fewer impurities you
will get.  When the lysis buffer (detergent) comes close
to the cell, it captures the lipids and the
Spooling the DNA
proteins – breaks open the cell destroying the
 If you have an acceptable amount of DNA, it fatty membrane  DNA is now released
can be “spooled” by rotating your colleting
Use of the Meat tenderizer
tool and then transferred into a clean tube /
container  Cuts the protein and DNA
 If you are careful you maybe able to wind up
 The tenderizer cuts the proteins away from
the DNA around a glass rod or a skewer.
the DNA
Position the tip of the glass rod where you can
see the threads of DNA. Steadily twist the rod  The tenderizer acts as an enzyme to cut
as if you were making cotton candy. Don’t go proteins
too quickly.
Use of the alcohol
What is that stringy stuff?
 Alcohol is less dense than water, so it floats
 DNA is a long, stringy molecule. on top.
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  Look for clumps of white stringy stuff where
the water and alcohol layers meet.
 DNA is not soluble in alcohol – other cell parts
are
 By adding alcohol, DNA precipitates out of the
solution and collect at the interface of the
alcohol and soap layer
 The colder the alcohol, the less soluble the
DNA will be
Why cold alcohol?
 DNA dissolves in water but precipitates in
alcohol
 Cold alcohol is used to separate DNA out of
water-based solutions
 This allows the DNA to be purified for
subsequent genetic testing
 Adding alcohol to a solution containing DNA
is a simple way to obtain the pure DNA
 Colder temperatures slow down enzymes that
can break down DNA, giving better extraction
results

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