Biology HW

May30PracticingLabSkills201905.pdf

Learning Goals

• Measure small volumes accurately using micropipettes • Perform dilution calculations • Use a spectrophotometer to get absorbance values for solutions • Create standard curves by hand and with Excel • Quantify target molecules with a standard curve • Accurately and completely describe the methods you choose in scientific journal style

By the end of lab today, you will be able to:

PRACTICING LAB SKILLS-JUST DO IT! May 30

Learning Goals

By the end of lab today, you will be able to:

Light Source

Entrance slit

Dispersion Device

Exit slit

Cuvette with sample

Detector

Understanding the Spectrophotometer (Turn on your spec now.)

If you pass a beam of orange-red (625 nm) light through each sample, which would have the LOWEST absorption?

A B C D

Hint: Remember, if something looks green, that means it REFLECTS green and ABSORBS everything else.

From left to right, cuvettes contain increasing concentrations of green dye.

Getting comfortable w/ concentrations

1. Which grid has the highest concentration of dots?

2. What are the concentrations of the grids?

3. On Grid A, which box (small or large) has the highest concentration of dots?

Getting comfortable w/dilutions

= 0.9 ml of water

= 0.1 ml

Your new solution is ____ as concentrated as the original solution.

Your new solution is ____ as dilute as the original solution.

Getting comfortable w/dilutions • Turn to the “Making Dilutions” section (pg. 24) in your manual and answer the

questions. Use the formula C1V1=C2V2 to help.

• Hint: You can use the formula C1V1 = C2V2 to solve this problem. • I) Your original solution had a concentration (C1) ____ g/L. • II) Your new solution has a concentration (C2) of ____ g/L in a volume of ____L (V2). • III) You will need to add ____ L (V1) of your original stock solution plus _____ L of water to make 100 ml of

the new solution.

y = 0.2755x + 0.0562 R² = 0.9977

0.5

1.5

2.5

0 1 2 3 4 5 6 7 8

M ea

n Ab

so rb

an ce

a t 6

25 nm

Concentration of Fast Green (ug/ml)

Standard Curve

Standard Curves • Absorbance values increase with increasing concentrations of dye.

Standard Curves • Today, you will be making a standard curve using methylene blue dye.

• You will need to mix the correct amounts of water and stock solution to create the concentrations needed for your standard curve.

• Fill in the table on page 25 of your lab manual. • Use can the formula C1V1=C2V2.

How to use micropipettes

Link to video

http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/cell-culture-videos/the-micropipette.html

Setting Micropipette Volumes

Follow the directions in your manual to complete the Micropipetting Practice Activity

Remember:

• Adjust the volume of the micropipette using the volume adjustment knob, not the plunger button

• Depress the plunger button to the first stop when aspiring liquids-not the second

• Submerge the disposable tip just below the surface of the liquid when aspirating

Standard Curves • Set up tubes for you standard curve and measure the absorbance values

using the instructions starting on pg. 25 of the lab manual.

“Blanking” a Spectrophotometer

• Why is a “blank” used with a spectrophotometer?

• A blank is used to eliminate background absorbance from your sample caused by the cuvette and your reagents.

• What should be used as a “blank”?

• A blank should contain everything that you have have in your sample except what you are measuring.

Estimating Unknown Concentrations • Measure absorbance values for “unknowns”

• Use your hand-sketched standard curve (p. 27) to estimate the concentration.

Compare the curves. What could have gone wrong for student 2?

0 1 2 3 4 5 6

A bs

or be

nc e

60 0

concentration (mg/mL)

0 1 2 3 4 5 6

A bs

or be

nc e

60 0

concentration (mg/mL)

Standard curve from Student 1 Standard curve from Student 2

Calculating Unknown Concentrations • Plot your standard curve in Excel

• Instructions are found in Appendix C of your lab manual.

• Use the equation of the line to calculate the concentrations of your unknowns. • Recall: y = mx + b

• y = absorbance value from spectrophotometer • m = the slope of the line • x = concentration of your unknown • b = y-intercept

CLEAN UP!! • Dispose of solutions in the appropriate waste containers.

• Wash tubes thoroughly with water and place the tubes upside down in the rack to dry.

• Dispose of used tips, tubes, or kimwipes in the trash.

• Wipe down benches.

• Wash hands.

SpeakWrite Approach

Writing an Introduction

Using Scientific Literature Part B-Introduction • Find a primary scientific article to be used as a source of background

information for your Biofuels Introduction • All groupmates should use a different primary article • Paraphrase the article

The Eberly Writing Studio • Consider having someone read it over who is not in the

class

• The Eberly Writing Studio

• Dial 304-293-5788 to schedule an appointment or stop by G02 Colson Hall to see if a tutor is available.

• Appointments can also be made online • http://speakwrite.wvu.edu/writing-studio

http://speakwrite.wvu.edu/writing-studio