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Lab Report Bio462

The document contains details of four experiments involving dilution calculations, protein determination assays, enzyme activity assays, and gel electrophoresis. It provides procedures, questions, and data related to each experiment for calculating concentrations, plotting standard curves, determining protein and enzyme levels, and explaining electrophoresis techniques.

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fuyuki miharu
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© © All Rights Reserved
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Download as DOCX, PDF, TXT or read online on Scribd
350 views

Lab Report Bio462

The document contains details of four experiments involving dilution calculations, protein determination assays, enzyme activity assays, and gel electrophoresis. It provides procedures, questions, and data related to each experiment for calculating concentrations, plotting standard curves, determining protein and enzyme levels, and explaining electrophoresis techniques.

Uploaded by

fuyuki miharu
Copyright
© © All Rights Reserved
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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EXPERIMENT 1: DILUTION AND MOLARITY

DILUTION PROBLEMS

i. How would you prepare:


a) 10 mL of 1:10 dilution of a 1 M NaCl solution and what would be the final
concentration?
b) 80 mL of a 1:20 dilution of a 1M NaCl ?
c) 50 mL of 1:25 dilution of a 1M NaCl ?

ii. How would you prepare exactly 6 mL of a 1:20 dilution (assume the concentration of
starting solution is “1”)?

iii. You are provided with an antibody solution (Ab) that has a concentration of 600 μg/ μL
for lab, it is necessary to make following dilution.
a) 10 μL of 600 μg/ μL Ab + 190 μL of buffer to make a 1: 20 dilution at ………
μg/ μL
b) 20 μL of 1:20 Ab + 40 μL of buffer to make a 1: 60 dilution at ……… μg/ μL
c) 5 μL of 1:60 Ab + 5 μL of buffer to make a ……. dilution at ……… μg/ μL
d) 10 μL of 1:60 Ab + 90 μL of buffer to make a ……. dilution at ……… μg/ μL
e) 10 μL of 1:60 Ab + 40 μL of buffer to make a ……. dilution at ……… μg/ μL
f) 10 μL of 1:60 Ab + 10 μL of buffer to make a ……. dilution at ……… μg/ μL

iv. How much 2.0 M NaCl solution would you need to make 250mL of 0.15 m NaCl
solution?

v. What would be the concentration of solution made by diluting 45.0mL of 4.2 M KOH to
250 mL ?

vi. What would be the concentration of a solution made by adding 250 mL of water to 45.0
mLof 4.2 M KOH?

vii. How much 0.20 M glucose solution can be made from 50.0 mL of 0.50 M of solution

viii. What is the molarity of a solution that has 4.5 mol of solute dissolved in 300 mL of
solution?
ix. What is the molarity of a solution of NaOH that has 0.491 g dissolved in 400 mL of
solution?
x. What is the molarity of solution prepared by diluting 10.00mLof 4.281 M solution to
50.00 mL ?

EXPERIMENT 2: PROTEIN DETERMINATION


QUESTIONS
1. Name the dye used in Bradford Assay (1 mark).

2. State the color change that occurs when proteins combine with the dye reagent (1 mark).

3. In Lowry protein assay, name the bond in protein that binds to copper ions (1 mark).

4. A Bradford assay was conducted to determine the total protein concentration in a sample.
A volume of 2 μL of the original sample was diluted to 100 μL with buffer before
performing the assay. The diluted sample gave an absorbance at 595 nm of 0.255. Using
the data for the standards below,

Protein concentration
A595
(μg/mL)
25 0.008
125 0.087
250 0.113
500 0.197
750 0.295
1000 0.429
1500 0.608

a) Plot a standard curve based on the data given (3 marks).


b) Determine the concentration of protein in the diluted sample (2 marks).
c) Calculate the total protein concentration in the original sample (2 marks).
EXPERIMENT 3: DETERMINATION OF ENZYMES ACTIVITY
QUESTIONS

1. Substitute the solution in the video into the equation below. (1 mark)
E + S E-S P

2. State the function of the Folin & Ciocalteau’s in the video. (1 marks)

3. In an enzymatic reaction, 1.0 mL of acetylcholinesterase was reacted with 2.0 mL of


acetylthiocholine iodide. 2.0mL of phosphate buffer was then been added in. Incubation for 10
minutes took place before 1.0 mL of acetic acid and 1.0 mL of dithiobisnitrobenzoic acid was
added up to the mixture. The mixture was further incubated for 10 minutes and 1.0 ml of the
sample was placed in cuvette for absorbance reading at 405 nm.
Using the data for the product standards below,

Absorbance Choline
(405 nm) (μmol)
0.00 0
0.067 0.05
0.113 0.1
0.197 0.2
0.395 0.4
0.729 0.8

a) Plot a standard curve based on the data given. (3 marks)


b) Determine the activity of the enzyme if the absorbance of enzymatic activity gave a
reading of 0.552 (3 marks).
c) Calculate the specific enzyme activity if the total protein content is 10 mg/mL and the
enzyme has been diluted 5X before enzymatic reaction takes place.(2 marks)
EXPERIMENT 4: GEL ELECTROPHORESIS

QUESTION

1. Define gel electrophoresis. (2 marks)

2. Describe how 1 % agarose gel was prepared. (3 marks)

3. State the number of wells in the agarose gel as shown in the first video. (1 mark)

4. State the function of ethidium bromide. (1 mark)

5. State the role of DNA ladder. (1 mark)

6. Identify which part of DNA molecule contain negative charge. (1 mark)

7. Explain the function of restriction enzyme. (1 mark)


ANSWERS

1. Gel electrophoresis is a method that used in molecular biology. It separate a mixed


population of macromolecules such as DNA in a matrix of agarose. The separation is based
on the molecular size and the molecule are separated because it being pushed by electrical
field across the gel.

2. The preparation step are as follow:

6. Pour the agarose solution in


1. Weight 1 g of agarose in the electrophrosis tank that
conical flask have been arranged properly
with border and comb

2. Add TAE or TBE buffer 5. Add ethidium bromide


in the sae conical flask into the agarose solution and
shake well

3. Heat up the flask in


microwave until all 4. Let the flask cool by
agarose are diluted. stirring with magnetic stir

3. 3 wells

4. Ethidium bromide is added in order to visualize the DNA in agarose gel electrophoresis
experiment

5. DNA ladder act as a references or standard to estimate the size of unknown DNA molecule
that are separated through the agarose gel.

6. The phosphate bone of DNA are negative charge.

7. The function of restriction enzyme is to cleave the DNA at restriction sites in order to create
smaller genetic fragments that can be separated and characterized by gel electrophoresis.

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