1. Lab Safety

2. Introduction 1.A chemical lab is potentially hazardous environment 2.Accident and injury can happen anytime 3.Lab safety is everyone’s responsibility 4.Lab safety standards and practices must be strictly followed

3. General Safety Rules 1. Listen to or read instructions carefully before attempting to do anything. 2.Always use appropriate personal protective equipments (lab coat, safety goggles, masks, gloves, no open shoes, no eye lenses)

4. General Safety Rules 3.After handling chemicals, always wash your hands with soap and water. 4. During lab work, keep your hands away from your face. 5.Tie back long hair. 6.Notify your supervisor if any spills or accidents occur.

5. General Safety Rules 7. Roll up loose sleeves. 8. Know the location of the fire extinguisher, fire blanket, eyewash station, and first aid kit. 9. Keep your work area uncluttered. Take to the lab station only what is necessary.

6. General Safety Rules 10. It is suggested that you wear glasses rather than contact lenses. 11. Never put anything into your mouth during a lab experiment. 12. Clean up your lab area at the conclusion of the laboratory period. 13. Never “horse around” or play practical jokes in the laboratory.

7. Laboratory Equipment zNever use any laboratory equipment unless you are trained & have been authorised to do so zAs well as injuring yourself you may cause very costly damage

8. Chemical Safety 1. Wear protective goggles and a lab apron whenever heating or pouring hazardous chemicals. 2. Never mix chemicals together unless you are told to do so (and then only in the manner specified). 3. Never taste any chemicals (you should never taste anything in the lab).

9. Chemical Safety 6. Follow the instructions of your teacher when disposing of all chemicals. 7.Wash your hands after handling hazardous chemicals. 8.Never mouth pippette

10. Electrical Safety 1. Lay electrical cords where no one can trip on them or get caught in them. 2. Be sure your hands and your lab area are dry before using electrical equipment. 3. Never poke anything into electrical outlets.

11. Electrical Safety 4. Unplug cords by pulling the plug and not the cord. 5. Unplug all electrical equipment at the end of the lab period.

13. Fire Extinguisher

14. Learn how to be always safe 1.Learn emergency procedures, and be familiar with the location of fire exits, fire extinguishers, blankets, water showers, eye fountains and first aid 2.Report all accidents, injuries and spills to your supervisor 3.Report any and all signs and symptoms of exposure to your supervisor

15. Biological safety 1.All biological samples are considered potentially infectious 2.Should be handled and processed using strict precautions

16. Waste Disposal 1.For disposal of contaminated waste, use containers with with yellow plastic garbage bags 2.Regular waste like paper etc go in the containers with black/white plastic bags 3.All sharp objects such as needles, scalpels and even broken glassware go in the yellow-red sharps container

17. Two choices !! or

18. DNA Extraction and Purification

19. Lab Equipments (To be used in this experiment) Automatic pipettes Microcentrifuge Vortex Water bath UV-spectrophotometer

20. Spectrophotometer Most of visible spectrophotometers are composed of: y Light source which works with visible wavelengths (400-700 nm) yMonochromator filter for choosing desired wavelength ySample holder (cuvette) yDetector yMeter or recorder

21. DNA Extraction zPrinciple: 1. Lysis of nucleated cells 2. Removal of contaminants: Any substance other than DNA, e.g., proteins 3. Measurement: UV absorbance at 260nm and 280nm Purity of DNA solution: 260/280 ratio DNA concentration: Absorbance at 260nm

22. DNA Extraction z Steps: y Lysis: of nucleated cells using lysis buffer y Binding: of DNA to the membrane of spin column y Wash: using wash buffer y Elution of pure DNA

23. Steps for DNA Extraction A. Lysis 1.Pipette 20 μl Qiagen protease into the bottom of a tube. 2.Add 200 μl whole blood sample. 3.Add 200 μl buffer AL and mix by pulse-vortex for 15 sec. 4.Incubate at 56 °C for 10 min. 5.Brief centrifuge to remove drops from the inside of the lid. 6.Add 200 μl ethanol (96 –100 %), mix by pulse-vortex for 15 sec. 7.Brief centrifuge to remove drops from the inside of the lid. B. Binding 8.Carefully apply the mixture from step 7 to the QIAamp spin column (in a 2 ml collection tube) without wetting the rim, close the cap. 9.Centrifuge at 6000 x g (8000 rpm) for 1 min. 10.Place the spin column in a clean 2 ml collection tube and discard the tube containing the filtrate.

24. Steps for DNA Extraction C. Wash 11.Carefully open the column and add 500 μl buffer AW1 close the cap. 12.Centrifuge at 6000 x g (8000 rpm) for 1 min. 13.Place the spin column in a clean 2 ml tube and discard the tube containing the filtrate. 14.Carefully open the column and add 500 μl buffer AW2 without wetting the rim, close the cap. 15.Centrifuge at 20,000 x g (14,000 rpm) for 3 min. 16.Place the spin column in a clean 1.5 ml microcentrifuge tube and discard the tube containing the filtrate. D. Elution of pure DNA 17.Add 200 μl elution buffer AE and incubate at room temperature for 5 min 18.Centrifuge at 6000 xg (8000 rpm) for 1 min. 19.Save the eluant (pure DNA) and discard the column.

25. Measurements yDilute the sample of purified DNA: Add 400 μL of AE to the purified DNA yMeasure the Absorbance at 260nm y Measure the Absorbance at 280nm y Assess the DNA purity: 260/280 ratio (Accepted ratio: 1.7 - 1.9)  Calculate DNA Conc.: Provided A260 = 1.0, DNA is 50 μg /ml, unknown DNA conc. can be calculated by cross multiplication A260 = 1.0 DNA conc. = 50 μg /ml A260 = 0.5 DNA conc. ? xCalculate DNA Yield: DNA Volume x DNA conc. Done by the spectrophotometer

26. DNA Yield: y If you have Volume of DNA solution: 200 μ l (0.2 ml) DNA conc.: 30 μg /ml Then the yield ( μg ) = Volume x Conc. 0.2 ml x 30 μg /ml = 6.0 μ g

27. DNA Applications z Purified DNA can be used for: 1. Molecular diagnosis of diseases (e.g., sickle cell anemia) 2. Forensic applications (e.g., paternity testing) 3. Molecular biology research

28. DNA Applications CONT’D Molecular techniques using purified DNA: a. Amplification techniques: polymerase chain reaction (PCR) b. Southern blotting c. Restriction Fragment length polymorphism (RFLP)