2. LABORATORY 1: TOOLS OF THE TRADE LSSI Alum, 2009 Jennifer Muñoz, Del Mar Union School District

3. www.amgenbiotechexperience.com Safety General Lab Safety Guidelines Use laboratory coats, safety glasses and gloves as appropriate Avoid restrictive clothing and open-toed shoes No eating or drinking in the lab Make sure that students are familiar with the operating instructions and safety precautions before they use any of the lab equipment Check all MSDS (Material Safety Data Sheets) for all chemicals and reagents in the lab before preparing and running the lab Wash hands at the conclusion of the lab. Lab Safety Guidelines for lab 1 Use caution with hot liquids and glassware. Wear heat-proof gloves to move hot glassware and liquids Make sure the power is turned off on power supplies before connecting electrodes After gel run is complete, turn off power supply then unplug electrodes If the power is on, do not touch the buffer or electrophoresis equipment as you may receive an electrical shock Never leave the electrophoresis power unit on without supervision. There is a risk of fire if the buffer leaks out or if the buffer should evaporate completely during electrophoresis

4. www.amgenbiotechexperience.com More on Lab Safety http://viewpure.com/xJG0ir9nDtc Tie back long hair Lab notebooks only Latex allergies for future labs

5. www.amgenbiotechexperience.com Curriculum & Standards ABE Manuals: Student and Teacher guide Online www.amgenbiotechexperience.comwww.amgenbiotechexperience.com Review pages in Teacher Guide: pages OV-5 through OV-12. Review Student Guide: Read pages 7 and 8.

6. www.amgenbiotechexperience.com Lab Prep & Aliquouting Guidelines Reagents/SuppliesAliquotStorage TempNotes 12 mls of Red Dye (RD)/kit1 ml/groupRTPlease return tubes 6 L of 1x SB buffer/kit RTPlease return Bottles 6 mls Solution 1 (S1).5ml/groupRTPlease return tubes 6 mls Solution 2 (S2).5ml/groupRTPlease return tubes 6 mls Solution 3 (S3).5ml/groupRTPlease return tubes Micropipette Practice Sheet 11/kitN/ARTPlease return sheets Round Practice Plates 12/kit Each plate will have ~15 wells. Each student should practice pipetting into one well. N/A4o4o Please return used and unused plates 10 gels per class. If you prefer to have your students pour their own gels, please let us know when you make your reservation on LARS. N/A4o4o Please return any unused gels. Discard used gels in the trash. Equipment/Supplies 10 Student boxes with the following: 1 p20 micropipette 1 microfuge rack 1 p200 micropipette 1 bag of microfuge tubes 1 p1000 micropipette 1 bag of microfuge tubes 1 waste and 1 box of refillable tips (2 ul-200 ul) 1 ice bucket 4 Mini centrifuges 10 Gel Electrophoresis chambers 10 Gel trays 10 gel combs 5 Power supplies

7. www.amgenbiotechexperience.com Making 1X SB Buffer Teacher Guide, page OV-30, Solution Preparation Use the formula C 1 V 1 = C 2 V 2 to determine the volume of stock solution (V 1 ) of the stock concentration (C 1 ). You will need to dilute with dH 2 O to prepare the final volume of working solution (V 2 ) at the working concentration (C 2 ).

8. www.amgenbiotechexperience.com What is Genetic Engineering? Students read pages 7 & 8. Introduce Diabetes storyline pages 9 through 15. Science Talk or Think-Pair-Share Accountable + Productive Refer to text Share with group Use academic vocabulary Rigorous and engaging at a deeper level.

9. www.amgenbiotechexperience.com Tools of the trade Micropipettes are used to measure and transfer very small volumes of liquids. Student guide pages 19 through 23

10. www.amgenbiotechexperience.com Video 1Video 2 Video 1Video 2 Using the Micropipette

11. www.amgenbiotechexperience.com Types of Micropipettes Lock

12. Parts of the micropipette

13. Reading a P-20 micropipette

14. Reading a micropipette (cont.) 0 5 0 0 5 0 0 5 0 5 μL50 μL500 μL P-20P-200P-2000

15. Micropipette use 1.Twist dial to desired volume 2.Add disposable pipette tip 3.Press plunger to first stop 4.To retrieve liquid, slowly release plunger 5.To transfer liquid, press plunger past first stop to second stop 6.Keep plunger down as you remove pipette 7.Eject tip

16. Pipetting Technique Hold the micropipette and microfuge tubes at eye level when loading or dispensing samples

17. Tips If you are pipetting, you should be holding the tube. Common mistake!

18. Critical Micropipetting Rules NEVER…use without a tip in place NEVER…lay it down with sample in the tip NEVER…let the “plunger” button snap back Laboratory practice: Turn to page 21: Review the materials section and follow steps 1 thru 15 in the methods section.

19. Practice Pipetting Sheet 20 μL 0 15 μL 0 10 μL 0 5 μL 0 2 μL 0

20. www.amgenbiotechexperience.com Making and Pouring a Gel: Follow the SOP

21. www.amgenbiotechexperience.com At first it will look cloudy…

22. www.amgenbiotechexperience.com Place in the microwave

23. www.amgenbiotechexperience.com It is ready to pour when it is cool to the touch… The solution should become clear…

24. www.amgenbiotechexperience.com Pour the agarose and let it solidify Remove the comb after the gel has solidified and lower the sides of the gel tray

25. Lab 1.2 Gel Electrophoresis Loading Gels Insert pipette tip: Under buffer level Above gel well

26. With both elbows on the table, use your other hand to stabilize the bottom of the pipette.

27. Different pipetting techniques – stability is the key K. Schramm

28. Tip should be above, not in the well. Do NOT punch the tip through the gel. Dye spreading under the well = punctured well Common Loading Errors

29. Proper Loading of the Gel Tip in buffer, above well Sample in well K. Schramm

30. www.amgenbiotechexperience.com A A 1 1 2 2 3 3 Which dye do you predict has the largest molecular weight?

31. Lab 1.2 Results Lab 1.2 Results K. Schramm Orange G (yellow), mol. Wt. = 452.4 Bromophenol blue (purple) = 691.9 Xylene cyanole (blue) = 538.6

32. www.amgenbiotechexperience.com A A Bromophenol blue (purple) is more negatively charged than xylene cyanole (blue) due to negatively charged bromine ions. Therefore, it travels farther than the smaller xylene cyanole. 1 1 2 2 3 3