DNA extraction presentation

DNA
EXTRACTION
Grade 10 Science / Life and Living: DNA
Compiled by Madre’ Nortje

SCIENCE @ WORK
EXPERIMENT 2.1

DNA EXTRACTION
A CONTEXTUAL APPROACH SCIENCE 10
(HEINEMANN): P125

2 GRADE 10: Life and Living
DATE of Practical: 17 March 2011

QUESTION:
WHAT DOES DNA STAND FOR?

DEOXYRIBONUCLEIC ACID

THE COMPLEX CHEMICAL COMPOUND FOUND INN
CHROMOSOMES THAT CONTAINS THE GENETIC
CODE

3
TODAY WE WILL CONNECT
DNA WITH:......extraction......

DO ONLY LIVING ORGANISMS
CONTAIN DNA?
What do you think?
Discuss in pairs for 1 minute
Give reasons.

EXTRACTING DNA FROM ANY
LIVING THING
 Just follow these 3 easy steps:

 Detergent
eNzymes
 Alcohol

5
 http://learn.genetics.utah.edu/content/labs/extraction/howto/

•All living organisms contain DNA in
their cells – FOUND in nucleus.

DNA - Deoxyribonucleic acid!
The nucleus is a membrane bound

YOU MEAN I CAN SEE IT? HOW?
structure that contains the cell's hereditary
information and controls the cell's growth
and reproduction. It is commonly the most
prominent organelle in the cell.

www..kamibudaksains.blogspot.com
•DNA - The complex chemical
compound found in chromosomes that 6

contains the genetic code.

FIGURE: ://
HTTP . . / / / 331/
EMPLOYEES CSBSJU EDU HJAKUBOWSKI CLASSES CH DNA/OLDNASTRUCTURE.HTML

PACKAGING OF DNA IN THE NUCLEUS

7

http://www.youtube.com/watch?v=tmVP1c
otozc

8

 DNA extraction refers to the process that scientists use to break down a piece of
evidence, such as a piece of hair or a drop of blood, in order to determine the
individual's DNA, which is unique to each person. Understand the process of DNA
extraction with information from a biology teacher in this free video on science.
Expert: Janice Crenetti
Contact: WeAreHDTV.com
Bio: Janice Creneti has a Bachelor of Science in secondary science education and
a Bachelor of Art in biology from Boston University.
Filmmaker: Christopher Rokosz
 Category:
 Entertainment
 Tags:
 science
 dna
 dna testing
 dna structure
 dna model
 dna replication
 dna molecule 9

REVIEWING OF INFORMATION ON:
LABORATORY SAFETY RULES AND RISK
ASSESSMENTS
 Do not enter the laboratory unless you are with a teacher.
 Never touch equipment in the laboratory unless you are told to use it.
 Don’t eat or drink in the laboratory.
 Always walk—never run.
 Wear protective clothing— a laboratory coat or apron and, when
appropriate, safety glasses. Never taste anything.
 Tie up long hair.
 Always point test tubes away from people.
 Check with your teacher on how to dispose of waste liquids and solids.
Broken glass should be cleaned up using gloves, a brush and dustpan,
and placed in a special bin.
 If you spill something on your skin or clothes, wash it immediately with
lots of water. Tell your teacher.
 Report all accidents and breakages to your teacher.
 After heating equipment, let it cool on a heatproof mat before picking it
up. This will avoid burns.
 Clean all equipment after use and put it back where you got it from. 10
Clean and dry your work bench.

RISK ASSESSMENT ON EXPERIMENT AND
INVESTIGATION – KIWI FRUIT

 Eyewear and protective clothing
 No eat or drinking
 Cover the MSDS’ e on Isopropanol
and Methylene blue – hard copy
handouts
(health and safety regulation)
 Any broken glassware must be
reported
 Care must be taken when using and
11
washing the blender – sharp blades

ISOPROPANOL – MSDS HAND OUTS
CHEMWATCH ISSUE DATE: 15/05/2010
 ISOPROPANOL - FLAMMABLE
 Dangerous Good 3
 SAFETY-DATA(tm) Ratings (Provided here for your convenience)
-----------------------------------------------------------------------------------------------------------
Health Rating: 2 - Moderate
Flammability Rating: 3 - Severe (Flammable)
Reactivity Rating: 2 - Moderate
Contact Rating: 3 - Severe
Lab Protective Equip: GOGGLES & SHIELD; LAB COAT & APRON; VENT HOOD; PROPER GLOVES; CLASS
B EXTINGUISHER
Storage Colour Code: Red (Flammable)
 Potential Health Effects
----------------------------------
Inhalation:
Inhalation of vapours irritates the respiratory tract. Exposure to high concentrations has a narcotic effect,
producing symptoms of dizziness, drowsiness, headache, staggering, unconsciousness and possibly death.
Ingestion:
Can cause drowsiness, unconsciousness, and death. Gastrointestinal pain, cramps, nausea, vomiting, and
diarrhoea may also result. The single lethal dose for a human adult = about 250 mls (8 ounces).
Skin Contact:
May cause irritation with redness and pain. May be absorbed through the skin with possible systemic effects.
Eye Contact:
Vapours cause eye irritation. Splashes cause severe irritation, possible corneal burns and eye damage.
Chronic Exposure:
Chronic exposure may cause skin effects.
Aggravation of Pre-existing Conditions:
Persons with pre-existing skin disorders or impaired liver, kidney, or pulmonary function may be more susceptible
to the effects of this agent

12

METHYLENE BLUE – MSDS HAND OUTS
CHEMWATCH ISSUE DATE: 15/05/2010
 METHYLENE BLUE
 SAFETY-DATA(tm) Ratings (Provided here for your convenience)
-----------------------------------------------------------------------------------------------------------
Health Rating: 2 - Moderate
Flammability Rating: 1 - Slight
Reactivity Rating: 1 - Slight
Contact Rating: 1 - Slight
Lab Protective Equip: GOGGLES; LAB COAT; VENT HOOD; PROPER GLOVES
Storage Colour Code: Green (General Storage)
-----------------------------------------------------------------------------------------------------------
Potential Health Effects
----------------------------------
This material is relatively nonhazardous in routine industrial situations.
Inhalation:
No adverse health effects expected from inhalation. May cause a short period of rapid or difficult breathing.
Ingestion:
A burning sensation of the mouth may be noted following ingestion of methylene blue. May cause nausea,
vomiting, diarrhea, and gastritis. Large doses may cause abdominal and chest pain, headache, profuse sweating,
mental confusion, painful maturation, and methemoglobinemia.
Skin Contact:
Not expected to be a health hazard from skin exposure. Methylene blue may colour the skin a bluish colour. May
cause photosensitization.
Eye Contact:
No adverse effects expected. May cause mechanical irritation.
Chronic Exposure:
No information found.
Aggravation of Pre-existing Conditions:
No information found.
13

NEW WORDS TO WORD BANK
ADD THESE TO YOUR EXCEL ALPHA TABLE
WORDS MEANING:

 Precipitate
 Spooling
 Denaturising
 Lysing
 Homogenized
 Aggregate
 Lipid
 Centrifuging
 PCR
14

•DO YOU THINK YOU HAVE VERY MUCH IN
COMMON WITH A KIWI FRUIT?

BELIEVE IT OR NOT, A KIWI'S GENETIC
MATERIAL IS VERY SIMILAR TO YOUR
OWN!

SEE AND TOUCH THE GENETIC MATERIAL
THAT YOU'LL EXTRACT FROM THE CELLS
OF A KIWI FRUIT.

15 Activity Overview
Extract DNA from kiwi fruit using simple household
chemicals

16

FRUIT
DNA EXTRACTION USING KIWI
HTTP://LEARN.GENETICS.UTAH.EDU/CONTENT/LABS/EXTRACTION
/HOWTO/

 MATERIALS / EQUIPMENT www.exploratorium.edu

TO EXTRACT DNA FROM THE CELLS OF KIWI FRUIT.!
AIM
 (per student group takes 30 minutes)

 ➤ ½ cup Kiwi fruit
 ➤ 200 mL water
 ➤ dishwashing detergent
 ➤ dropping pipette
 ➤ fine mesh kitchen strainer & cheese cloth
 ➤ glass rod
 ➤ large beaker
 ➤ large test tube
 ➤ light microscope
 ➤ meat tenderiser
 ➤ methylene blue
 ➤ microscope lamp
 ➤inoculation needle
 ➤ microscope slide and cover slip
 ➤ paper towelling
 ➤ small beaker of alcohol (Isopropanol)
 ➤ spatula
17
 ➤ test-tube rack
 ➤ mortar and pestle

PART A:
EXTRACTING THE DNA
TAKE NOTES – NO TEXTBOOKS ALLOWED
WHEN PERFORMING EXPERIMENT –
MUST RECALL PROCEDURES / METHOD

 METHOD:

 1. Peal and Place the kiwi and
water in the beaker and mush it with a mortar
and pestle.
 DNA SOURCE About 125 ml / 1/2 cup
 Twice as much water as DNA source (250
ml/ 1 cup)
 Table salt, large pinch 1g / 1/4 teaspoon
 Stir until the mixture is of a thin, soupy
18
consistency.

WHAT MUST I DO NOW?

DISHWASHING
STRAIN THE DNA MIXTURE
DETERGENT

19

 2.Pour the thin cell mixture through the
kitchen strainer (cheesecloth) into another
large beaker.

 3.Measure 12 ml of the soup into a small
beaker
 Add 2 ml of liquid detergent

 Swirl to mix stir thoroughly using a glass
rod
 Let mixture sit in container with hot tap
water (60 - 65°C) for 5 - 10 minutes
20

WHAT MUST I DO NOW?
MEAT TENDERIZER
ALCOHOL SEPARATION
ENZYME POWDER
 www.forums.overclockers.com.au

21

 4. Add a pinch of enzyme (meat
tenderizer) to mixture.
 (Using pineapple juice or contact lens
cleaning solution will do the same as
tenderizer.)
 Gently stir with toothpick/ skewer for 5
minutes, continue stirring, not too
vigorously. Be careful! If you stir too hard,
you'll break up the DNA, making it harder to
see.

 5. Quarter-fill a large test tube with the
22
mixture.

 SLOWLY pour the same amount ice -
cold (70 - 95% isopropyl or ethyl alcohol)
into test tube pour alcohol down the side
of the test tube.
 Tilting the test tube will make this easier to
do.

 It
forms a layer on top of the cell mixture.
 DO NOT MIX THE TWO LAYERS
TOGETHER.
 Amount of alcohol and mixture should
be the same.
23

SEPARATION USING ALCOHOL!

24

 7. Observe the mixture for a few minutes.
You will see a white, threadlike substance
rise from the mixture to rest above in the
alcohol layer.
 This is the DNA that you have extracted
from the cells of the kiwi fruit.

 8. You can get more DNA to precipitate from
the solution using a DNA collecting tool
(glass or paper clip hook or cut inoculation
needle)
 Gently lift the water solution up into the
alcohol layer (this allows more DNA to get in
25
contact with the alcohol and precipitate).

 INFORMATION:
 DNA precipitates as a white stringy /
―snotty‖ film at the water - alcohol interface
and eventually will rise into the alcohol layer
from the mixture layer.

 Allow test tube to sit for several minutes.
 The clearer the DNA is the fewer impurities
you have.

 Ifyou have an acceptable amount of DNA, it
can be "spooled" by rotating your collecting
tool and then transferred into a clean tube /
container. 26

WOW – SPOOLING THE DNA!!!
 If you are careful you may be able wind up the DNA
around a glass rod or a skewer. Position the tip of the
glass rod or skewer where you can see the threads of
DNA. Steadily twist the rod or skewer as if you were
making candy floss. Alternatively use a straw to pull it
out by suction – be careful not to get in you mouth.

 Don’t go too quickly.

 You should be able to pull the strands of DNA out of the
mixture.

 ASK STUDENTS TO TAKE MOBILE PHONE
PICTURES OF THEIR OWN EXTRACTED DNA AND
COMPARE APPEARANCE WITH OTHERS
 Photos of steps can be inserted in flow
diagram.(ICT) 27

 Ifyou want to save your DNA, you can
transfer it to a small container filled
with alcohol
 Leave tube / container uncapped until
the ethanol has evaporated.

 DNA can be stored in the fridge (dry or
water /buffer can be added).
 You can now investigate the property.
29

WHAT IS THAT STRINGY STUFF?
DNA IS A LONG, STRINGY MOLECULE.
THE SALT THAT YOU ADDED IN STEP ONE HELPS
IT STICK TOGETHER. SO WHAT YOU SEE ARE
CLUMPS OF TANGLED DNA MOLECULES!

DNA NORMALLY STAYS DISSOLVED IN WATER,
BUT WHEN SALTY DNA COMES IN CONTACT WITH
ALCOHOL IT BECOMES UNDISSOLVED. THIS IS
30
CALLED PRECIPITATION. THE PHYSICAL FORCE
OF THE DNA CLUMPING TOGETHER AS IT
PRECIPITATES PULLS MORE STRANDS ALONG
WITH IT AS IT RISES INTO THE ALCOHOL.

THE WHY’S!!!!
 Blending separated the pea cells.
 In order to extract DNA from a cell, the associated membranes
and proteins must first be removed (break apart the cells) and
then physically separated (loosen the tough cell wall) from the
DNA.
 To see the DNA, we have to break open these two sacks.
 We do this with detergent and salt( Sodium can be involved in
several of the steps ).
 Sodium is an element. It's chemical symbol is Na for Natrium,
the Latin word for sodium. It is a positive ion and often
associates with negative ions as part of useful compounds.
 Salt help precipitate protein and carbohydrates away from the
DNA.

 Salt helps strip away the proteins associated with DNA. + and
– charge

31

THE WHY’S!!!!
 Salt and Detergents are used to break down cell
walls and nuclear membranes to release the DNA.
 They work by chemically poking holes in the cell
membranes or walls.
 Once holes are poked in the membranes, the
membranes can be further disrupted mechanically,
as with a blender.
 After that, it is easier to get the contents of the cell
out, including the DNA.

 LETS HAVE A LOOK AT THE CELL!
32

!
http://learn.genetics.utah.edu/content/labs/extraction/howto/
HOW TO EXTRACT DNA FROM ANYTHING LIVING
CELL TO DNA

33

THE WHY’S!!!! WHY DETERGENT?
 Each cell is surrounded by a sack (the cell
membrane)
 DNA is found inside the second sack (nucleus)
within each cell.
 To see the DNA we have to break it open.

 A cell membrane has 2 layers of lipid(fat) molecules
with protein going through them.
 When the lysis buffer (detergent) comes close to
the cell, it captures the lipids and the proteins -
breaks open the cell destroying the fatty membrane
- DNA now released into the solution. 34

A CELL'S MEMBRANES HAVE TWO LAYERS OF
LIPID (FAT) MOLECULES WITH PROTEINS GOING
THROUGH THEM.

35

 Why detergent? How does detergent work?

Think about why you use soap to wash dishes or
your hands. To remove grease and dirt, right?
 Soap molecules and grease molecules are made of
two parts:

 1.(Blue) Heads, which like water.
 2.(Green) Tails, which hate water.
36

AFTER ADDING THE DETERGENT, WHAT DO
YOU HAVE IN YOUR SOUP?

37

THE WHY’S!!!! ENZYME
(MEAT TENDERIZER)
 The DNA in the nucleus of the cell is molded,
folded, and protected by proteins. The tenderizer
cut the proteins away from the DNA
 In this experiment, meat tenderizer acts as an
enzyme to cut proteins just like a pair of scissors.
 The meat tenderizer cuts the proteins away from
the DNA

38

THE WHY’S!!!!
CUTTING PROTEIN AND DNA
The DNA in the
nucleus of the cell is
moulded, folded, and
protected by proteins

39

THE WHY’S!!!!ALCOHOL
 Alcohol is less dense than water, so it floats on top.
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 in it.
40

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
required, and colder temperatures slow
down enzymes that can break down
DNA, giving better extraction results.
41

WHY IS DNA EXTRACTION IMPORTANT?
 DNA extraction is an important molecular biology
procedure.

 By definition, extraction is taking DNA out of any
type of cell for the purpose of analysis.

 See Handouts for more information on the
use of DNA extracted (extension only)

42

SUMMARIZE
DNA EXTRACTING
FROM KIWI FRUIT
43

BREAK DOWN CELL WALLS
 Cells walls have to be destroyed to reach the
DNA within cells.

 Extraction procedures to obtain pure DNA have to
get rid of all the molecular and chemical
components of the tissue from which the DNA is
being extracted.

 First steps involve lysing or destroying the cell
walls. This can be done with a variety of chemical
agents that are caustic to cell membranes but do
not harm the DNA. Cells can also be sonicated,
homogenized, or ground up to destroy membranes.
44

REMOVING
 Removing Lipids
 Once the cell walls have been destroyed, a
detergent is added to get rid of the fats and oils
that make up the cell membranes. Detergents
cause the fats and oils to dissolve into the
solution.
 Remove Proteins

 Proteins and enzymes can be digested by
adding a protease to the solution. Proteases
break down proteins into small peptides and
amino acids.
45

PRECIPITATING AND PURIFYING
 Precipitate DNA
 Adding cold alcohol to a solution will cause DNA
to precipitate and aggregate. The DNA can be
collected by centrifuging the sample and pouring
off the liquid layer. The DNA should exist as a
small pellet in the bottom of the centrifuge tube.
 Purify DNA
 The DNA can be washed by re-suspending it in a
cold alcohol solution and re-centrifuging it
several times to obtain a very pure DNA sample.
Typical alcohols used to precipitate DNA include
ethanol and Isopropanol. This process leaves a
very pure sample for stringent DNA testing.
46

PART B: OBSERVING
EXTRACTED DNA UNDER THE
MICROSCOPE

47

PART B: A CLOSER LOOK
 8. Use a dropping pipette to carefully remove some of the
threadlike substance from the top of your preparation.

 9. Place one or two drops onto the middle of a microscope
slide.

 10. Add two drops of methylene blue. Wait 3 or 4 minutes to
allow the methylene blue to be absorbed by the DNA.

 11. Carefully place a cover slip on the slide. Gently press a
folded piece of paper towelling over the top of the prepared
slide to soak up any excess liquid.

 12. Observe the DNA under low power, then high power.

48

DISCUSSION
ANSWER IN GROUP RELATED WORKED.

 1 Write a detailed description of the material
floating at the top of the test tube after the alcohol
was added.

 2 Describe the DNA as it appears under the
microscope under high power.

 3 Prepare a diagram of your DNA specimen.

 4 What was the reason for using methylene blue?
49

HOMEWORK:
CONSTRUCT A FLOW CHART / DRAWING/ MIND MAP
OF THE PROCESS YOU USED EXTRACTING THE DNA
FROM KIWI’S

 Next to each step explain how the method you used
was important. (Hint: What substances make up the
membranes of cells and cell organelles?

 How do detergents work?

 What is the active ingredient in meat tenderiser?

50

TIPS:
STEPS FOR FLOW CHART
 In order to release the DNA from the nuclei
of the pea cells you first separated the cells
from one another.
 Then the cell and nuclear membranes
needed to be ruptured to release the cell
contents and the contents of the nucleus.
 Once removed from the nuclear membrane
the DNA had to be untangled into the
visible, threadlike structures you ended up
with. 51

HOMEWORK:
EXTENSION GROUP QUESTIONS
 Where can DNA be found in the cell?
 Discuss the action of the soap (detergent) on the cell. What is
the purpose of the soap in this activity?
 What was the purpose of the Sodium Chloride? Include a
discussion of polarity and charged particles.
 Why was the cold ethanol added to the soap and salt mixture?
 Describe the appearance of your final product?
 Draw a diagram of DNA containing 5 sets of nucleotide bases
labelling the hydrogen bonds between the bases.
 References and Resources: Adapted from Berry Full of DNA
by Diane Sweeney for Biology: Exploring Life to be published
by Prentice Hall. Websites:
http://www.carlinvilleschools.net/linke/Biology/DNA.htm
http://carnegieinstitution.org/first_light_case/horn/DNA/dnaind
ex.html
52

HTTP://WWW.GENOMEBC.CA/EDUCATION/TEACHERS/CLASSROOM-ACTIVITIES/DNA-EXTRACTION/

53

EXTRA:
CHROMOSOMES EXIST IN PAIRS
 Because our chromosomes exist in pairs (and consequently
we have 2 alleles of each gene), we are a diploid species. This
is why our somatic cells are represented as "2n". Our gametes
(sperm and ova), on the other hand, are haploid, and are
represented as "n". Other species may have different ploidy, for
example:
 triploid (3n): seedless watermelons www.cyberus.ca
 tetaploid (4n): salmonidae fish
 pentaploid (5n): Kenai birch
 hexaploid (6n): some types of wheat, kiwi fruit
 octaploid (8n): acipenser (a genus
of sturgeon fish, strawberies)
 decaploid (10n): some strawberries
 dodecaploid (12n): some types of amphibians, e.g. Xenopus 54
ruwenzoriensis

YOUR EXTRACTED DNA UNDER THE
MICROSCOPE LOOKED LIKE .........!

 Collaboration-Brainstorming on what to look at
under the
 - DNA extraction- DNA is too small for even a
microscope to see and after the extraction it just
appears like a blob. True or false?
 Could you any DNA strand?

 www.employees.csbsju.edu

55

HANDOUT ON DNA EXTRACTION:
BACKGROUND EVERY DAY LIFE
EXTRA READING MATERIAL
(EXTENSION )
 DISCOVERING DNA
 DNA ON THE INSIDE

 USE OF DNA EXTRACTION INN EVERYDAY LIFE

 WHY IS DNA TESTING GOOD?

 MOLECULAR BIOLOGY

 PCR-based diagnostics of genomic DNA

56

REFERENCES:
 http://www.squidoo.com/how-to-get-dna-from-a-kiwi-fruit
 Why Is DNA Testing Good? | eHow.com http://www.ehow.com/about_6684410_dna-
testing-good_.html#ixzz1MkbcIJs5
 Why Is DNA Extraction Important? | eHow.com http://www.ehow.com/list_5839095_dna-
extraction-important_.html#ixzz1MkZDrqyY
 Why Is Sodium Used in DNA Extraction? | eHow.com
http://www.ehow.com/about_6504902_sodium-used-dna-
extraction_.html#ixzz1MkaGWg57
 Why Is Cold Alcohol Used in DNA Tests? | eHow.com
http://www.ehow.com/about_6399349_cold-alcohol-used-dna-
tests_.html#ixzz1MkbEmEdu
 http://learn.genetics.utah.edu/content/labs/extraction/howto/
 www.. kamibudaksains.blogspot.com

 http://www.chemwatch gold.com
 References and Resources: Adapted from Berry Full of DNA by Diane Sweeney for
Biology: Exploring Life to be published by Prentice Hall. Websites:
http://www.carlinvilleschools.net/linke/Biology/DNA.htm 57
http://carnegieinstitution.org/first_light_case/horn/DNA/dnaindex.html

REFERENCES:
 Uses of DNA Extraction | eHow.com
http://www.ehow.com/about_5344428_uses-dna-
extraction.html#ixzz1MkWQB2TZ
 Science 10: A Contextual Approach Heineman
Queensland Science Projects : Regan Spence
Maggie Spenceley
 http://en.wikipedia.org/wiki/Molecular_biology
 http://www.clinchem.org/cgi/content/extract/44/10/2
201
 http://employees.csbsju.edu/hjakubowski/classes/c
h331/dna/oldnastructure.html
 http://www.genomebc.ca/education/teachers/classr
58
oom-activities/dna-extraction/

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