BIO113

CUMULATIVE PRACTICAL
SPRING 2024

Name__________________________________ Date____________

By signing this exam, I pledge on my honor that I have done this work to the best of my abilities, with
honor and integrity, without giving or receiving unauthorized assistance.

Signature___________________________________

1. Use the line graph and the given equation to answer the following questions.

a. What is the protein concentration (mg/ml) of a tissue homogenate sample with an

absorbance of 0.85 OD @ 595 nm? (Show your work)
b. You now want to dilute the tissue homogenate sample (from above) to a final
concentration of 0.5 mg/ml. Assuming you want to make 10 ml of the diluted
sample, what volume (ul) of homogenate is needed? What volume of water is needed?
(Show your work).

SHOW WORK IN BOX BELOW

FINAL ANSWERS GO IN THE CHART BELOW

Volume of tissue homogenate (ul)

Volume of water (ul)

2. Answer the following questions regarding our experiments assessing succinate
dehydrogenase (SDH) activity in tissue homogenates:

a. How is DCIP used to assess the activity of this enzyme?

b. What happens to DCIP in the presence of Malonate? Will the absorbance be high or low
@ 600 nm?

c. What can be a negative control reaction/condition in this experiment to confirm that

changes to DCIP is due to SDH and not another enzyme?
3. This semester we performed a colorimetric acid to quantify the amount of glucose in
our bovine tissue homogenate samples. Answer the following related questions to this
specific lab.

A. In reaction 2, What is B? (An enzyme, Protein, Dye) ________________________

B. Fill in the chart below:

RESULTS: COLOR Rank the ABSORBANCE @

(Colorless, red, dark red) 520 nm (1=lowest; 2= highest)

1. Homogenate sample +
amyloglucosidase

2.Homogenate sample -
amyloglucosidase

C. What is the purpose of amyloglucosidase?

D. If the concentration for condition 1 (see table in B) was 1.5 mg/ml and the concentration from

condition 2, was 0.5 mg/ml, answer the following:

a. What is the total glucose? _________________________

b. Estimate the amount of glucose from glycogen in the homogenate _________________

A. What was the purpose of using a bicarbonate indicator and algal beads this semester? What
processes were we assessing/comparing?

B. What is the relationship between CO2 and the color of the bicarbonate indicator?

4. Imagine you performed tonicity experiments with sheep red blood cells. In one set of
experiments, you determined the isotonic concentration of NaCl and in another you
determined the isotonic concentration of Sucrose. How do you expect the isotonic
concentrations of the two to vary? Use a quantitative comparison to describe this
difference.
5. Draw a line between the correct temperatures to each step of PCR, and give the
definition

94°C extension:

72°C denature:

60°C annealing:

6. The image below is a picture of a pBADCherry plasmid. This plasmid contains a red
fluorescent gene, mCherry.
a. Fill the table below based on your predictions about growth and the fluorescent ability of
E.coli cells transformed with this plasmid.

PLATES/CONDITIONS GROWTH RED FLUROESENCE

(YES OR NOW) (YES OR NO)

LB - pBADCherry

LB + pBADCherry

LB – pBADCherry + amp

LB + pBADCherry + amp

LB – pBADCherry + ara

LB + pBADCherry + ara

LB – pBADCherry + amp + ara

LB + pBADCherry + amp + ara

b. Imagine you extracted DNA from a E.coli colony on your LB + pBADCherry plate,
performed a PCR amplifying a 1,000 bp product for the cherry gene. Draw your gel
predictions below in LANE 1.

In, LANE 2, draw your predictions for a PCR reaction where we failed to add primers.

In LANE 3, draw your predictions for a PCR reaction with DNA from a E.coli colony from
a LB + pBADCherry + amp + ara plate.
 7. DEMONSTRATION:
Now, using the appropriate micropipette, answer the following questions

a. Find the tube labeled “dye” and pipette 200 ml of dye into 800 ml of water.
**** (Must get approved by your instructor and or TA for credit) ****

b. Then, measure the absorbance @ 595 of the provided blank ______________

c. Measure the absorbance @ 595 of the dilution you generated. ______________

EXTRA CREDIT

SDS vs Agarose electrophoresis

Questions SDS PAGE Agarose

What type of Biomacromolecule it

detects?

Is it ran horizontally or vertically?

What dye or stain is used to visualize

biomacromolecules?

What are the charges of the molecules

being separated? (+ or -)