Protocol of the DNA Extraction in Kitchen

©SIProp Project, 2006-2008 1

Protocol of the DNA Extraction in KitchenNoritsuna [email protected]


What’s DNA?Ｄ NA is a Blueprint for Living Being(Ex.Human).

Weight: 0.003ng = 0.000000000003gChromosome is a Packet for DNA.

Human has 46 Chromosomes(2 Sets of 23 Kinds of Chromosome).

Nucleus is a Core of Cell.Cell is minimum Unit of Living Being.

Human has 37 trillion(37,000,000,000,000) Cells.

Source: http://www.ashg.org/education/everyone_1.shtml


Let’s Extract DNA from Banana


SummaryTotal Time: 20-30mins

Protocol1. Make 10% Saline of 30mL.2. Cut Banana to 20g.3. Put Saline, Banana, Dishwashing liquid(6

drops) & Contact lens cleaning solution(6 drops) in Plastic Bag.

4. Mash Banana & Mix them in Plastic Bag. (3mins)

5. Filter “4” Solution in Test Tube. (3mins)6. Make a micro Tube of DNA Solution7. Mix 95% Ethanol & Filtered Solution in Test

Tube.1. Pick up Banana’s DNA from Test Tube.


Check ToolsPipette

Pipette Chip

Dishwashing Liquid

Plastic Bag

Scales Beaker

Test Tube Micro Tube

Tube Stand

Funnel &Filter Paper

Lens cleanin

g solution

Ethanol

Salt


1-1, Make 10% Saline of 30mLRequired Tools:

ScalesBeakersSpoonMuddlerSalt

CAUTION: Please Skip this part & Use Distilled Water(30mL),if you do the DNA Test Experiment.


1-2, Scale Salt & WaterWhat is “10% Saline of 30mL”?

10% Saline = 90% Water + 10% Salt30mL = 90% Water + 10% Salt = 27mL Water + 3g Salt

Today’s Experiment is Not a Strict Experiment.≒ 30mL Water + 5g Salt

Because it is easy to scale them!


1-3, Mix Salt & WaterUse a Plastic Beaker & a Muddler

Target Transparency → → →


2-1, Cut Banana to 20g.Required Tools:

ScalesCutting BoardKnifeBanana


2-2, Cut Banana to 20g.Peel & Cut(Slice) Banana.

Point: Cut Thin!


3-1, Put All Items in the Plastic Bag

Mixed Items:Saline: 30mLCut Banana: 20gDishwashing liquid: 6 DropsContact lens cleaning solution: 6 Drops

Required Tools:Plastic Bag


3-2, Put All Items in the Plastic Bag1. Put Cut Banana2. Put Saline3. Drop 6 Drops of Dishwashing liquid4. Drop 6 Drops of Contact lens cleaning

solution


3-3, Close the Plastic Bag


4-1, Mash Banana & Mix them in Bag.

Required Tools:Your Hands!


4-2, Mash BananaMash Banana for 3mins


5-1, Filter “4” Solution in Test Tube

Required Tools:FunnelFilter PaperTest TubeTube Stand


5-2, Fold Filter Paper1. Fold Crescent-shape2. Fold Quadrant of a Circle3. Make Cone-shape (Open the Filter

Paper)


Easy Way of Filtering Mashed Something

Use Gauze Sheets as 1st Filter & a Coffee Filter as 2nd Filter.

Gauze(1st Filter) prevents clogging of a Filter Pater/Coffee Filter with Mashed Banana.A Coffee Filter is easier to buy a Paper Filter.


5-3, Set Up Funnel & Test Tube1. Set The Cone in the Funnel

2. Set the Test Tube into the Tube Stand3. Insert the Funnel to the Test Tube


5-4, Drip the Mashed Banana Solution1. Move the Solution to the One Side of

the Bag2. Cut the Bag of Opposite Side

3. Pour the Solution into the Funnel4. Wait for 3mins


5-5, Check the Amount of Solution

Pick up the Funnel & the Test Tube togetherCheck the Amount of Filtered Solution

DNA melts into this solution.Minimum Solution


Try to Make one more Tube!


6-1, Make a micro Tube of DNA Solution

Required Tools:Micro Pipette (200uL or 1000uL)Pipette ChipsMicro Tube (0.2mL=200uL) aka PCR Tube


6-2, Set Pipette’s AmountWe need “100uL” Liquid.

Set Scale Dial at “100(200uL)” or “010(1000uL)”

Amount Dial


6-3, Set Up Pipette Chip Insert Pipette Chip to Tip of the Pipette


6-4, How to Use the Pipette?Push Button has 2 kinds of type.

1st Stop ・・・ Suck Operation2nd Stop ・・・ Flash Operation

Procedure1. Push & Keep the Push Button to 1st Stop2. Insert the Pipette Chip into the Solution3. Release the Push Button Slowly

1. Suck the Solution with the Pipette.4. Extract the Pipette from the Test Tube5. Insert the Pipette Chip into a micro Tube6. Push & Keep the Push Button to 1st Stop

1. & Keep this position 1sec7. Push & Keep the Push Button to 2nd Stop(Flash)8. Extract the Pipette from the micro Tube

Push Button


6-5, Move the Solution to a micro Tube

Move the Solution from the Test Tube to a micro Tube

Suck the Filtered Solution from the Test TubeProcedure1. Push & Keep the Push Button to 1st Stop2. Insert the Pipette Chip into the Solution of the Test

Tube3. Release the Push Button Slowly

1. Suck the Filtered Solution with the Pipette.4. Extract the Pipette from the Test Tube


6-6, Move the Solution to a micro Tube

Move the Solution from the Test Tube to a micro Tube

Move the Filtered Solution to the micro TubeProcedure5. Insert the Pipette Chip into a micro Tube6. Push & Keep the Push Button to 1st Stop

1. & Keep this position 1sec7. Push & Keep the Push Button to 2nd Stop(Flash)8. Extract the Pipette from the micro Tube


6-7, Throw the Pipette ChipPush the Eject Pipette Chip Button on Garbage.

Please Change the Pipette Chip by each Using Pipette.

Eject Pipette

Chip Button


6-8, Why made it?This Tube is used in DNA Test Phase.For PCR(Polymerase Chain Reaction) Sequence

Amplify a single copy or a few copies of a piece of DNA.

https://en.wikipedia.org/wiki/Polymerase_chain_reaction




Please Stop this Experiment, if you do the DNA Test

Experiment.

And Go to “Protocol of the DNA Test in Kitchen”


7-1, Mix Ethanol & the Filtered Solution

Required Tools:90% over Ethanol (1-2 Times Amount of the Filtered Solution)The Tubes of the Filtered Solution

The Test TubeThe micro Tube


7-2, Put Ethanol into the Test Tube

Using the Funnel without a Filter1-2 Times Amount of the Filtered Solution


7-3, Pick Up DNA1. Insert a Fork into the Test Tube2. Stir this Solution Super Slowly by the

Fork1. A White Haze gradually appears.

3. Pick Up this White Haze1. This is Banana’s DNA!!!


7-4, The micro Tube SideMix Ethanol(100uL)

Put Ethanol into the micro Tube by Pipette


7-5, Flick the micro Tube with a Finger

When Flick a finger, A White Haze appears.

This is called “Tapping Method” in Bio Science


The Principle of This Experiment


The Principle of SurfactantDishwashing Liquid

This alias is Surfactant. This is a catalyst which can mix oil with water.

Surfactanthttps://en.wikipedia.org/wiki/Surfactant

Cell & Cell nuclear have membrane. A structure of Cell Membrane & Nuclear Membrane is Lipid(≓oil) Bilayer.

Lipid Bilayerhttps://en.wikipedia.org/wiki/Lipid_bilayer

Dishwashing Liquid can break down Cell Membrane & Nuclear Membrane.

Source: https://en.wikipedia.org/wiki/Cell_(biology)#/media/File:Wilson1900Fig2.jpg

https://en.wikipedia.org/wiki/Surfactant

https://en.wikipedia.org/wiki/Surfactant

https://en.wikipedia.org/wiki/Lipid_bilayer

https://en.wikipedia.org/wiki/Lipid_bilayer


The Principle of ProteaseContact lens cleaning solution

One of the ingredients of this is protein-degrading enzymes(=Protease).

Proteasehttps://en.wikipedia.org/wiki/Protease

Chromosome is made by DNA & some kinds of Proteins.

Contact lens cleaning solution can separate Chromosome into DNA & some kinds of Proteins.

https://en.wikipedia.org/wiki/Protease

https://en.wikipedia.org/wiki/Protease


The Principle of FilterFilter

DNA is called Nucleic Acids.Nucleic Acids melts into water.

Your Hand

Surfactant

Protease Water


The Principle of IonizationSaline(Salt)

Salt alias is Sodium Chloride.Chemical Formula of Sodium Chloride: NaCl

Saline alias is Electrolyte Solution of NaCl(= NaCl is ionized to Na+ & Cl- in water)

Ionic Formula of Sodium Chloride: NaCl→ Na+ + Cl-

DNA IonizationOne part of the DNA backbone is a phosphate group.

Red Circle Part is ionized to P-O-(Ionized DNA) & H+ in water.

Ionic Formula : DNA→ H+ + P-O-(Ionized DNA)DNA Ionization Status is Negative(-).

Repulsive force acts on each Ionized DNA.Ionized DNA can NOT fit a lump.Source: http://www.chemguide.co.uk/organicprops/aminoacids/dna1.html#top


The Principle of Ionic BondIonic Bond

Between Positive(+) Ion & Negative(-) Ion bond.

Positive(+): Na+ from SaltNegative(-): P-O-(Ionized DNA) from DNA

Ionic Formula : Na+ + P-O-(Ionized DNA) → P-ONa = DNA Natrium

DNA Natrium doesn’t have Electric Charge. DNA Natrium CAN fit a lump.


The Principle of Precipitation95% Ethanol

When Solution of the Filtered Solution & Ethanol are mixed, DNA Natrium moves the Filtered Solution(Water) side to Ethanol side.

Solubility: the Filtered Solution(Water) > EthanolDNA Natrium Lump is precipitated in Ethanol.


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Protocol of the DNA Extraction in Kitchen

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