2. LABORATORY 1: TOOLS OF THE TRADE
LSSI Alum
Mary Haus, Chaffey Joint Union High School District

3. Curriculum & Standards
NGSS -
HS-LS Construct an explanation based on evidence for DNA, etc.
Science Practices - Asking Questions and Defining problems
DCI - LS1.A - Structure and Function (both)
DCI - LS1.C - Organization for Matter and Energy Flow in Organisms
DCI - LS3.A - Inheritance of Traits

4. Curriculum & Standards
Common Core English Language Arts -
RST : Synthesize information from a range of sources into a coherent understanding…
WHST : Draw evidence from informational texts to support analysis, reflection, and research.

5. Curriculum & Standards
21st Century Core Competencies -
Cognitive Domain - Cognitive Processes and Strategies:
Reasoning/argumentation
interpretation
Cognitive Domain - Knowledge:
Oral and written communication
Active listening
Interpersonal Domain - Teamwork and Collaboration:
Communication
Collaboration
Teamwork
Interpersonal Skills

6. Curriculum & Standards
Review pages in Teacher Guide: pages and 63
Review Student Guide: Read page 19

7. Lab Prep & Aliquouting Guidelines
Reagents/Supplies
Aliquot
Storage Temp
Notes
12 mls of Red Dye (RD)/kit
1 ml/group RT
Please return tubes
6 L of 1x SB buffer/kit
Please return Bottles
6 mls Solution 1 (S1)
.5ml/group
6 mls Solution 2 (S2)
6 mls Solution 3 (S3)
Micropipette Practice Sheet 11/kit
N/A
Please return sheets
Round Practice Plates 12/kit
Each plate will have ~15 wells. Each student should practice pipetting into one well. 4o
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.
Please return any unused gels.
Discard used gels in the trash.
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette microfuge rack
1 p200 micropipette bag of microfuge tubes
1 p1000 micropipette bag of microfuge tubes
1 waste and 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

8. 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

9. Lab Overview/Introduction
Teacher Manual – pgs 49 – 50
Student Manual – pg 19

10. Lab 1.1 – How to use a micropipette
Teacher Manual – Preparation on pgs 51 – 53
Student Manual – pgs
The Basic Laboratory Tool: Micropipette

11. An Introduction to Pipetting

12. Using a Micropipette Activity
Handout found on MATSC

13. Steps for using a Micropipette
Set the volume on the micropipette .
Use the micropipette to attach a pipette tip.
Pick up and hold the microcentrifuge tube containing the solution at eye-level
Push the plunger to the first stop.
Insert the pipette tip into the tube containing a solution.
Aspirate the solution by slowly releasing the plunger of the micropipette.
Dispense the solution by slowly pushing the plunger to the first stop and then to second stop.

14. Setting the Volume on a Micropipette Activity (found on MATSC)
1) The smallest amount of volume that I can
measure with a P-20 pipette is…
2) When I use a P-20 pipette the maximum volume I can measure is…

15. Remember, The Pipette can be damaged if you pass these…

16. Lab 1.1: How to use a micropipette23
Student Manual – pgs

17. ***Make sure to fill with 1X SB buffer before loading.
Lab 1.2 (part A)
Practice loading into gel.
Student manual pgs
***Make sure to fill with 1X SB buffer before loading.
Things separate by size AND by charge (size is the biggest factor).

18. See additional power point
Intro to Flowgramming
See additional power point

19. Learning a New Skill: Electrophoresis and Gel Analysis

20. Let’s Flowgram! Now on to the lab! In groups of 4
Use large whiteboards and markers
Student manual, pgs
Lab 1.2 part B
Lets compare :) - teacher manual pg. 61
Now on to the lab!

21. Lab 1.2 (Part B) Student Manual pgs. 26 - 32
Run three samples marked S1, S2, and S3. (part B)
Solution Tube 1 = blue + purple
SolutionTube 2 = blue + purple + yellow
SolutionTube 3 = blue
Yellow dye = orange G
Purple dye = bromophenol blue
Blue dye = xylene cyanole
Things separate by size AND by charge (size is the biggest factor).

22. Lab 1.2 (Part B)
Which dye do you predict has the largest molecular weight? A 3 2 1
Things separate by size AND by charge (size is the biggest factor).
Remember – Things separate by size AND by charge (size is the biggest factor)

23. Some GE tips… Gel box lids slide off, they do not pull off
Make sure that you pour enough buffer to completely cover the gel by about 2 mm. If you can see dimples in the buffer where the wells are you need to add more.
Wells should be on the black side so your samples can “RUN to RED.”
Place your gel box close enough to connect to power supply so that you do not need to move it to plug it in.

24. While Samples are running…
Gel electrophoresis Card Sort
Gel electrophoresis Activity
Lab 1.3

25. Gel Electrophoresis Card Sort

26. Gel Electrophoresis Activity

27. Gel Electrophoresis Activity

28. Lab 1.3 Optional Activity to demonstrate precision
Found in teacher manual, pgs and RM1 (last section of the manual)
Complete if time permits

29. Lab 1.2 – Results Orange G (yellow), mol. Wt. = 452.4
Bromophenol blue (purple) = 691.9
We only use negatively charged dyes because DNA is negatively charged. Note that there are many bromine groups (which are negatively charged) on Bromphenol blue.
Xylene cyanole (blue) = 538.6
We only use negatively charged dyes because DNA is negatively charged. Note that there are many bromine groups (which are negatively charged) on Bromphenol blue.
K. Schramm

30. Lab 1.2 Results
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. S1 S2 S3
Solution Tube 1 = blue + purple
SolutionTube 2 = blue + purple + yellow
SolutionTube 3 = blue
Don’t forget to get a photo of your gel!

31. Tips/Strategies/Questions
Preparation -
Pipette practice additional techniques – page covers, ziplocs
Results on overhead transparencies
Flowgrams on colored sheets by day
Who Dunnit? for different level classes

32. Contact Mary Haus Los Osos High School 6001 Milliken Ave.
Rancho Cucamonga, CA 91737