Lab # 1: Laboratory practices and Plant DNA extraction at home Practical Of Genetics BIOL 2121 Lab # 1: Laboratory practices and Plant DNA extraction at home 08/03/1440

Objectives: To introduce the student the Laboratory practices and safety used. To introduce the different micropipettes. To introduce the concepts and calculations for dilutions and solutions. Plant DNA extraction at home. 08/03/1440

1- Some of the labs. Materials are dangerous: 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. Methanol. Acetic acid glacial. Hydrochloric acid ( HCl ). Ultraviolet Transilluminators. Sodium hydroxide. Ethidium Bromide. 08/03/1440

(Micro Pipette,Gilson) Pipetman 2- To introduce the different micropipettes:                (Micro Pipette,Gilson) Pipetman Model 0.2 - 2 µl P2 0.5 - 10 µl P10 2 - 20 µl P20 20 - 100 µl P100 50 - 200 µl P200 200 - 1000 µl P1000 1000 - 5000 µl P5000 1 -10 ml P10ml 08/03/1440

08/03/1440

Symbol Name  Factor d deci 10-1 c centi 10-2 m milli 10-3 µ micro 10-6 n nano 10-9 p pico 10-12 f femto 10-15 a atto 10-18 z zepto 10-21 y yocto 10-24 08/03/1440

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. 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. 08/03/1440

Terminology and Concepts: Stock solution: concentrated solution which is being diluted Working solution: diluted solution, ready to use. Diluent: the fluid used for diluting concentrate. Aliquot: a measured sub-volume of original sample. Dilution factor (DF): ratio of final volume/aliquot volume (final volume = aliquot + diluent) Concentration factor (CF): ratio of aliquot volume divided by the final volume (inverse of the dilution factor) 08/03/1440

Example: What is the dilution factor if you add 0 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 dilution factor is equal to the final volume divided by the aliquot volume: 10 mL/0.1 mL = 1:100 dilution (102) The Concentration Factor for this problem = aliquot volume/final volume = 0.1/(0.1 + 9.9) = 0.01 or 10-2 concentration This is sometimes given as: C1 X V1 = C2 X V2 Remember: V2 > V1 C2 < C1 08/03/1440

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. 08/03/1440

Molarity : 08/03/1440

4- Plant DNA Extraction 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. The DNA found in strawberry and onion cells for example can be extracted using common, everyday materials. We will use an extraction buffer containing salt, to break up protein chains that bind around the nucleic acids, and dish soap to dissolve the lipid (fat) part of the strawberry cell wall and nuclear membrane. 08/03/1440

Cont. 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. 08/03/1440

Materials heavy plastic bag. strawberry & Onion bulb. DNA Extraction buffer (soapy, salty and water). Cheesecloth and funnel. 50mL vial / test tube. glass rod, inoculating loop, or popsicle stick. Ethanol 90%. 08/03/1440

Cont. Extraction buffer: detergent (dishwasher or shampoo). Detergent/salt solution: 20 ml detergent 20 g non-iodized salt 180 ml distilled water 08/03/1440

Procedure for Strawberry DNA extraction: Place one strawberry in plastic bag. Smash/grind up the strawberry using your fist and fingers for 2 minutes. Careful not to break the bag! Add 10mL of extraction buffer (salt and soap solution) to the bag. mush the strawberry in the bag again for 1 minute. Assemble your filtration apparatus as shown bellow. Pour the strawberry slurry into the filtration apparatus and let it drip directly into your test tube. 08/03/1440

Cont. Slowly pour cold ethanol into the tube. OBSERVE 5 minutes. Dip the loop or glass rod into the tube where the strawberry extract and ethanol layers come into contact with each other. OBSERVE 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. 08/03/1440

Image 08/03/1440

Good Luck 08/03/1440