General GeneticsGeneral Genetics

Lab. 1

Laboratory practices and Plant DNA extraction

The Islamic University

Faculty of Science

Biology and Biotechnology Department

غزة – االسالمية الجامعة

العلوم كلية

والتكنولوجيا االحياء قسمالحيوية

ObjectivesObjectives::

- - To introduce the student the Laboratory To introduce the student the Laboratory practices and safety used.practices and safety used.

- To introduce the different micropipettes.- To introduce the different micropipettes. - To introduce the concepts and - To introduce the concepts and

calculations for dilutions and solutions.calculations for dilutions and solutions. - Plant DNA extraction at home.- Plant DNA extraction at home.

All of the chemicals listed below are highly toxic and hazardous All of the chemicals listed below are highly toxic and hazardous compounds. Take extreme care when handling these compounds. compounds. Take extreme care when handling these compounds. On contact with skin/eyes wash immediately with water:On contact with skin/eyes wash immediately with water:

EthanolEthanol MethanolMethanol Acetic acid glacialAcetic acid glacial Hydrochloric acid ( HCl )Hydrochloric acid ( HCl ) Ultraviolet TransilluminatorsUltraviolet Transilluminators Sodium hydroxideSodium hydroxide Ethidium BromideEthidium Bromide

11 - -Some of the labs. Materials are Some of the labs. Materials are dangerousdangerous::

22 - -To introduce the different To introduce the different micropipettesmicropipettes::

               (Micro Pipette,Gilson) Pipetman

Model

P20.2 - 2 µl±0.024 - ±0.030 µl

P100.5 - 10 µl±0.025 - ±0.1 µl

P202 - 20 µl±0.1 - ±0.2 µl

P10020 - 100 µl±0.35 - ±0.8 µl

P20050 - 200 µl±0.5 - ±1.6 µl

P1000200 - 1000 µl±3 - ±8 µl

P50001000 - 5000 µl±12 - ±30 µl

P10ml1 -10 ml±30 - ±60 µl

FactorName Symbol

10-1decid

10-2centic

10-3millim

10-6microµ

10-9nanon

10-12picop

10-15femtof

10-18attoa

10-21zeptoz

10-24yoctoy

Micropipette TipsModel : Eppendorf

33 - -To introduce the concepts and calculations To introduce the concepts and calculations for dilutions and solutionsfor dilutions and solutions::

Preparing reagents and solutions is a never-Preparing reagents and solutions is a never-ending task in most laboratories. This is a basic ending task in most laboratories. This is a basic laboratory skill that often confuses people at laboratory skill that often confuses people at first. first.

Here we present the standard, general approach Here we present the standard, general approach to computing dilutions and concentrations; the to computing dilutions and concentrations; the Dilution Factor Technique. It is a convenient way Dilution Factor Technique. It is a convenient way of computing dilutions at the bench. Work of computing dilutions at the bench. Work through this section BEFORE coming to lab.through this section BEFORE coming to lab.

Terminology and Concepts:Terminology and Concepts:

Stock solutionStock solution:: concentrated solution which is being concentrated solution which is being diluteddiluted

Working solutionWorking solution:: diluted solution, ready to use diluted solution, ready to use DiluentDiluent:: the fluid used for diluting concentrate the fluid used for diluting concentrate AliquotAliquot:: a measured sub-volume of original sample. a measured sub-volume of original sample.

Dilution factor (DF)Dilution factor (DF):: ratio of final volume/aliquot volume ratio of final volume/aliquot volume (final volume = aliquot + diluent) (final volume = aliquot + diluent) Concentration factor (CF):Concentration factor (CF): ratio of aliquot volume ratio of aliquot volume

divided by the final volume (inverse of the dilution factor) divided by the final volume (inverse of the dilution factor)

Example:Example: What is the dilution factor if you add What is the dilution factor if you add 0.10.1 mLmL aliquot of a specimen to aliquot of a specimen to 9.9 mL9.9 mL of diluent of diluent ? ?

  The final volume is equal the the aliquot volume plus The final volume is equal the the aliquot volume plus the diluent volume:  the diluent volume:  0.1 mL + 9.9 mL = 10 mL0.1 mL + 9.9 mL = 10 mL

The dilution factor is equal to the final volume divided The dilution factor is equal to the final volume divided by the aliquot volume: by the aliquot volume: 10 mL/0.1 mL10 mL/0.1 mL = 1:100 dilution = 1:100 dilution (10(1022) )

The Concentration Factor for this problem = aliquot The Concentration Factor for this problem = aliquot volume/final volume = 0.1/(0.1 + 9.9) = 0.01 or 10volume/final volume = 0.1/(0.1 + 9.9) = 0.01 or 10-2-2

concentrationconcentration

C1 X V1 = C2 X V2C1 X V1 = C2 X V2

Concentrated stock solutions - using "X" Concentrated stock solutions - using "X" unitsunits::

Stock solutions of stable compounds are Stock solutions of stable compounds are routinely maintained in labs as more routinely maintained in labs as more concentrated solutions that can be diluted concentrated solutions that can be diluted to working strength when used in typical to working strength when used in typical applications. The usual working applications. The usual working concentration is denoted as 1X. A solution concentration is denoted as 1X. A solution 20 times more concentrated would be 20 times more concentrated would be denoted as 20X and would require a 1:20 denoted as 20X and would require a 1:20 dilution to restore the typical working dilution to restore the typical working concentration.concentration.

MolarityMolarity: :

44 - -Plant DNA ExtractionPlant DNA Extraction

1- 1- Plant DNA Extraction at home:Strawberry Plant DNA Extraction at home:Strawberry fruit and Onion bulb:fruit and Onion bulb:

Background: The long, thick fibers of DNA Background: The long, thick fibers of DNA store the information for the functioning of store the information for the functioning of the chemistry of life. DNA is present in the chemistry of life. DNA is present in every cell of plants and animals.every cell of plants and animals.

The DNA found in strawberry and onion The DNA found in strawberry and onion cells for example can be extracted using cells for example can be extracted using common, everyday materials.. common, everyday materials..

principleprinciple

We will use an extraction buffer containingWe will use an extraction buffer containing

11 - -salt, to break up protein chains that bind around salt, to break up protein chains that bind around the nucleic acidsthe nucleic acids , ,

22 - -dish soap to dissolve the lipid (fat) part of the dish soap to dissolve the lipid (fat) part of the strawberry cell wall and nuclear membranestrawberry cell wall and nuclear membrane . .

33--Alcohol is used to precipitate the DNA. Because Alcohol is used to precipitate the DNA. Because DNA is soluble in water, alcohol (ethanol) DNA is soluble in water, alcohol (ethanol) causes the DNA to precipitate and come out of causes the DNA to precipitate and come out of the solutionthe solution..

MaterialsMaterials heavy plastic bagheavy plastic bag strawberry & Onion bulbstrawberry & Onion bulb DNA Extraction buffer (soapy, salty and water)DNA Extraction buffer (soapy, salty and water) Cheesecloth and funnelCheesecloth and funnel 50mL vial / test tube50mL vial / test tube glass rod, inoculating loop, or popsicle stickglass rod, inoculating loop, or popsicle stick Ethanol 90%Ethanol 90%

Extraction buffer:Extraction buffer: detergent (dishwasher or shampoo) detergent (dishwasher or shampoo) 20 ml detergent 20 ml detergent 20 g non-iodized salt 20 g non-iodized salt 180 ml distilled water180 ml distilled water

Procedure for Strawberry DNA Procedure for Strawberry DNA extractionextraction::

1. Place one strawberry in plastic bag.1. Place one strawberry in plastic bag. 2. Smash/grind up the strawberry using your fist 2. Smash/grind up the strawberry using your fist

and fingersand fingers for 2 minutes. Careful not to break the bag!for 2 minutes. Careful not to break the bag! 3. Add 10mL of extraction buffer (salt and soap 3. Add 10mL of extraction buffer (salt and soap

solution) to the bag.solution) to the bag. 4. mush the strawberry in the bag again for 1 4. mush the strawberry in the bag again for 1

minute.minute. 5. Assemble your filtration apparatus as shown 5. Assemble your filtration apparatus as shown

bellow.bellow.

6. Pour the strawberry slurry into the filtration apparatus 6. Pour the strawberry slurry into the filtration apparatus and let it drip directly into your test tube.and let it drip directly into your test tube.

7. 7. SlowlySlowly pour cold ethanol into the tube. OBSERVE 5 pour cold ethanol into the tube. OBSERVE 5 minutes.minutes.

8. Dip the loop or glass rod into the tube where the 8. Dip the loop or glass rod into the tube where the strawberry extract and ethanol layers come into contact strawberry extract and ethanol layers come into contact with each other. OBSERVEwith each other. OBSERVE

9- extract a sample of DNA from the tube and place it on 9- extract a sample of DNA from the tube and place it on a clean  microscope slide; level the mass on the slide a clean  microscope slide; level the mass on the slide and stain it with a nuclear dye (ex: Toluidine, Methylene and stain it with a nuclear dye (ex: Toluidine, Methylene Blue, Aceto-Orcein; if necessary, add a little water and Blue, Aceto-Orcein; if necessary, add a little water and mount the coverslip.mount the coverslip.

DNA starting to precipitate in alcohol

5 min. CondensedDNA