1. General Genetics Lab. 1 Laboratory practices and Plant DNA extraction The Islamic University Faculty of Science Biology and Biotechnology Department الجامعة الاسلامية – غزة كلية العلوم قسم الاحياء والتكنولوجيا الحيوية

2. Objectives: - To introduce the student the Laboratory practices and safety used. - To introduce the student the Laboratory practices and safety used. - To introduce the different micropipettes. - To introduce the different micropipettes. - To introduce the concepts and calculations for dilutions and solutions. - To introduce the concepts and calculations for dilutions and solutions. - Plant DNA extraction at home. - Plant DNA extraction at home.

3. All of the chemicals listed below are highly toxic and hazardous compounds. Take extreme care when handling these compounds. On contact with skin/eyes wash immediately with water: All of the chemicals listed below are highly toxic and hazardous compounds. Take extreme care when handling these compounds. On contact with skin/eyes wash immediately with water: Ethanol Ethanol Methanol Methanol Acetic acid glacial Acetic acid glacial Hydrochloric acid ( HCl ) Hydrochloric acid ( HCl ) Ultraviolet Transilluminators Ultraviolet Transilluminators Sodium hydroxide Sodium hydroxide Ethidium Bromide Ethidium Bromide 1- Some of the labs. Materials are dangerous:

4. 2- To introduce the different micropipettes: (Micro Pipette,Gilson) Pipetman Model ±0.024 - ±0.030 µl0.2 - 2 µl P2 ±0.025 - ±0.1 µl0.5 - 10 µl P10 ±0.1 - ±0.2 µl2 - 20 µl P20 ±0.35 - ±0.8 µl20 - 100 µl P100 ±0.5 - ±1.6 µl50 - 200 µl P200 ±3 - ±8 µl200 - 1000 µl P1000 ±12 - ±30 µl1000 - 5000 µl P5000 ±30 - ±60 µl1 -10 ml P10ml

6. Symbol Name Factor ddeci10 -1 ccenti10 -2 mmilli10 -3 µ micro10 -6 nnano10 -9 ppico10 -12 ffemto10 -15 aatto10 -18 zzepto10 -21 yyocto10 -24 Micropipette Tips Model : Eppendorf

7. 3- To introduce the concepts and calculations for dilutions and solutions: Preparing reagents and solutions is a never- ending task in most laboratories. This is a basic laboratory skill that often confuses people at first. Preparing reagents and solutions is a never- ending task in most laboratories. This is a basic laboratory skill that often confuses people at first. Here we present the standard, general approach to computing dilutions and concentrations; the Dilution Factor Technique. It is a convenient way of computing dilutions at the bench. Work through this section BEFORE coming to lab. Here we present the standard, general approach to computing dilutions and concentrations; the Dilution Factor Technique. It is a convenient way of computing dilutions at the bench. Work through this section BEFORE coming to lab.

8. Terminology and Concepts: Stock solution: concentrated solution which is being diluted Stock solution: concentrated solution which is being diluted Working solution: diluted solution, ready to use Working solution: diluted solution, ready to use Diluent: the fluid used for diluting concentrate Diluent: the fluid used for diluting concentrate Aliquot: a measured sub-volume of original sample. Aliquot: a measured sub-volume of original sample. Dilution factor (DF): ratio of final volume/aliquot volume Dilution factor (DF): ratio of final volume/aliquot volume (final volume = aliquot + diluent) (final volume = aliquot + diluent) Concentration factor (CF): ratio of aliquot volume divided by the final volume (inverse of the dilution factor) Concentration factor (CF): ratio of aliquot volume divided by the final volume (inverse of the dilution factor)

9. Example: What is the dilution factor if you add 0.1 mL aliquot of a specimen to 9.9 mL of diluent? The final volume is equal the the aliquot volume plus the diluent volume: 0.1 mL + 9.9 mL = 10 mL The final volume is equal the the aliquot volume plus the diluent volume: 0.1 mL + 9.9 mL = 10 mL The dilution factor is equal to the final volume divided by the aliquot volume: 10 mL/0.1 mL = 1:100 dilution (10 2 ) The dilution factor is equal to the final volume divided by the aliquot volume: 10 mL/0.1 mL = 1:100 dilution (10 2 ) The Concentration Factor for this problem = aliquot volume/final volume = 0.1/(0.1 + 9.9) = 0.01 or 10 -2 concentration C1 X V1 = C2 X V2 C1 X V1 = C2 X V2

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

11. Molarity :

12. 4- Plant DNA Extraction 1- Plant DNA Extraction at home:Strawberry fruit and Onion bulb: Background: The long, thick fibers of DNA store the information for the functioning of the chemistry of life. DNA is present in every cell of plants and animals. Background: The long, thick fibers of DNA store the information for the functioning of the chemistry of life. DNA is present in every cell of plants and animals. The DNA found in strawberry and onion cells for example can be extracted using common, everyday materials.. The DNA found in strawberry and onion cells for example can be extracted using common, everyday materials..

13. principle We will use an extraction buffer containing 1- salt, to break up protein chains that bind around the nucleic acids, 2- dish soap to dissolve the lipid (fat) part of the strawberry cell wall and nuclear membrane. 3-Alcohol is used to precipitate the DNA. Because DNA is soluble in water, alcohol (ethanol) causes the DNA to precipitate and come out of the solution.

14. Materials heavy plastic bag heavy plastic bag strawberry & Onion bulb strawberry & Onion bulb DNA Extraction buffer (soapy, salty and water) DNA Extraction buffer (soapy, salty and water) Cheesecloth and funnel Cheesecloth and funnel 50mL vial / test tube 50mL vial / test tube glass rod, inoculating loop, or popsicle stick glass rod, inoculating loop, or popsicle stick Ethanol 90% Ethanol 90% Extraction buffer: detergent (dishwasher or shampoo) Extraction buffer: detergent (dishwasher or shampoo) 20 ml detergent 20 ml detergent 20 g non-iodized salt 20 g non-iodized salt 180 ml distilled water 180 ml distilled water

15. Procedure for Strawberry DNA extraction: 1. Place one strawberry in plastic bag. 1. Place one strawberry in plastic bag. 2. Smash/grind up the strawberry using your fist and fingers 2. Smash/grind up the strawberry using your fist and 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 solution) to the bag. 3. Add 10mL of extraction buffer (salt and soap solution) to the bag. 4. mush the strawberry in the bag again for 1 minute. 4. mush the strawberry in the bag again for 1 minute. 5. Assemble your filtration apparatus as shown bellow. 5. Assemble your filtration apparatus as shown bellow.

16. 6. Pour the strawberry slurry into the filtration apparatus and let it drip directly into your test tube. 6. Pour the strawberry slurry into the filtration apparatus and let it drip directly into your test tube. 7. Slowly pour cold ethanol into the tube. OBSERVE 5 minutes. 7. Slowly pour cold ethanol into the tube. OBSERVE 5 minutes. 8. Dip the loop or glass rod into the tube where the strawberry extract and ethanol layers come into contact with each other. OBSERVE 8. Dip the loop or glass rod into the tube where the strawberry extract and ethanol layers come into contact with each other. OBSERVE 9- extract a sample of DNA from the tube and place it on a clean microscope slide; level the mass on the slide and stain it with a nuclear dye (ex: Toluidine, Methylene Blue, Aceto-Orcein; if necessary, add a little water and mount the coverslip. 9- extract a sample of DNA from the tube and place it on a clean microscope slide; level the mass on the slide and stain it with a nuclear dye (ex: Toluidine, Methylene Blue, Aceto-Orcein; if necessary, add a little water and mount the coverslip.

17. DNA starting to precipitate in alcohol 5 min. Condensed DNA