MCB120L

AI-1

Practice Problems: Experiment 1

Consult as needed: Table 1.1 pKa values for buffers in dilute solution
1.1. What volume of glacial acetic acid (17.6 N) and weight of sodium acetate3 H2O (F.W. = 136) is
required to make 100 ml of 0.20 M buffer, pH 4.06? (Note: F.W. stands for formula weight in g/mol).
1.2. a) What volume of glacial acetic acid (17.6 N) and weight of sodium acetate3 H2O (F.W.= 136) is
required to make 100 ml of 0.20 M buffer, pH 5.06?
b) How is 100 ml of 0.050 M buffer, pH 5.06, prepared from a stock of 0.20 M buffer, pH 5.06?
1.3 What quantities of reagents are needed to make 250 ml of a 0.050 M acetate buffer at pH 5.5?
Available reagents: 1.0 N HCl, 1.0 N KOH, CH3COOK (FW 98.15, pKa 4.76)
1.4. a) What weights of sodium carbonate (FW = 106) and sodium bicarbonate (FW = 84) are required
to make 100 ml of 0.20 M buffer, pH. 10.38?
b) How is 100 ml of a 0.080 M buffer, pH 10.38, prepared from a stock of 0.20 M buffer, pH 10.38?
1.5. What volume of hydrochloric acid (11.7 N) and weight of Tris (MW = 121) are required to make
100 ml of 0.20 M buffer, pH 8.71?
1.6. What volume of concentrated HCl (11.7 N) and weight of Tris base (MW = 121 g/mole) is needed
to make 100 ml of 0.20 M buffer, pH 8.45?
1.7. a) Glycine has both weak acid (the primary amine) and weak base (the carboxyl group) properties,
making it a useful as a buffer. What are the pKas of glycines amino and carboxyl groups? What is
the structure of pure glycine in H2O?
b) What is the pH of 0.2M glycine in H2O?
c) If 50 mls of 0.20 M glycine is mixed with 50 mls of 0.1 M KOH, what is the resulting pH?
d) How will the pH change if the temperature is increased above 200C?
1.8. a) If 50 mls of 0.20 M glycine is mixed with 50 mls of 0.1M HCl, what is the resulting pH?
b) How will the pH change if the temperature is increased above 200C?
1.9. a) Phenol Red is a weakly acidic dye which is yellow in the HA form and red in the A form. What
concentration of Phenol Red is required to obtain an absorbance of 0.50 at 550 nm at pH 7.8?
Note: 550 of A = 2.5 x 104 M-1 cm-1; 550 of HA = 0; pKa = 7.8..
b) What would the absorbance of the solution in part (a) be if the pH was 8.8?

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1.10. Determine the pKa of a dye. The dye was diluted into several different buffers and the A450 was
measured (Table I). Using the data in Table I along with the 450 (anionic form) = 50,000 M-1 cm-1,
find the pKa of the dye.
Table I
Buffer

Dye stock volume,

ml
0.5

A450

0.01 M acetate, pH 4.5

Buffer volume,
ml
4.5

0.01 M HEPES, pH 7.0

4.5

0.5

0.32

0.01 M Tris, pH 8.0

4.5

0.5

0.81

0.01 M carbonate, pH 10.0

4.5

0.5

1.00

0.01 M NaOH

4.5

0.5

1.00

0.00

1.11. Calculate the pKa of m-ultra red when equimolar amounts of the pH indicator m-ultra red are
mixed in separate test tubes with large molar excesses of buffers at seven different pH values. All
buffer plus dye solutions were diluted to the same final volume and the absorbances at 550 nm were
read.
equimolar concentrations of
m-ultra red at various pH values
pH

A550

0.00

0.00

2
6
10

0.00
0.54
1.40

11
12

1.40
1.40

Challenge Problem
1.12. When an enzyme catalyzes the hydrolysis of X to Y, one mole of protons is released per mole of
substrate utilized. When this reaction was carried out at 24C in a 3 ml reaction mixture buffered only
by Tris buffer at an initial pH of 8.5, 20 moles of X were converted
to Y. What is the minimum concentration of Tris buffer required to prevent the pH from decreasing
below 8.3?

MCB120L

AI-3

Practice Problems: Experiment 2

2.1. What are the units of absorbance, A?
2.2. A 6.00 mg/l solution of adenine in water has an absorbance of 0.590 at 260 nm in a 1-cm cuvette.
Adenine has a molecular weight of 135.1. What is its molar extinction coefficient, , at 260 nm?
2.3. a). A 1.00 mg/ml solution of bovine serum albumin (BSA) in neutral phosphate buffer has an A260
of 0.600. If the molecular weight of BSA is 66,000 Daltons, what is its molar extinction coefficient?
b). A 1.00 mg/ml solution of bovine serum albumin (BSA) in neutral phosphate buffer has an A280
of 0.667. If the molecular weight of BSA is 66,000 Daltons, what is its molar extinction coefficient?
c). At what wavelength is the absorbance of the 1.00 mg/ml solution of BSA the most sensitive?
2.4. Calculate the molar absorption coefficient of NADH at 340 nm from the spectral data collected on a
16.5 mg/l solution of NADH (see figure below). Molecular weight of NADH = 665 g/mol.

2.5. For guanosine, 275 = 8400 M1 cm1 and 245 = 9150 M1 cm1 in 0.1 M HCl. In the same
solvent, tyrosine has an 275 = 1340 and 245 = 170. A mixture of these compounds in 0.1 M HCl has
an A275 = 0.69 and an A245 = 0.49. What are the molar concentrations of the two solutes?
2.6. One ml of a guanosine solution was mixed with 4 ml of 0.1 M HCl. When 0.07 ml of this solution
was added to 1.5 ml of 0.1 M HCl in a cuvette, the absorbance at 275 nm was 0.50. What was the
millimolar concentration of guanosine in the original solution? The appropriate molar extinction
coefficient is given in problem 2.5.

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2.7. A pure zinc-containing protein was found to have an absorption coefficient of 1.5 (mg/ml)1 cm1
at 280 nm. A concentrated solution of the protein in water was found to contain 7.2 g/ml zinc. A 0.1
ml aliquot of the same solution was diluted with 0.9 ml of water (dilution A) and 0.2 ml of dilution A
was added to 1.0 ml of water (dilution B). Dilution B had a measured A280 = 0.375. What are the
minimum molecular weight and minimum molar absorption coefficient of the protein? The atomic
weight of zinc is 65.4 g/mol. (Hint: minimum molecular weight assumes only one mole of Zn binds
to one mole of protein; so, finding the moles of Zn in the protein solution will give the moles of
protein).
2.8. A solution of unknown protein concentration was assayed by the Bradford protein assay, in 0.5 ml
assay volumes, in a 0.5 ml cuvette with a 1 cm path-length. Absorbances were read in a Shimadzu
spectrophotometer at 595 nm. Below are the data obtained from the assay. Fill in the table, plot the
standard curve, and determine the protein concentration from the data.
Bradford Protein Assay

tubes

BSA standard
or unknown sample

H 2O
l

reagent
l

average
A595

1,2

0 l 0.2 g/l BSA

250

250

0.151

3,4

10 l 0.2 g/l BSA

240

0.203

5,6

20 l 0.2 g/l BSA

230

0.258

7,8

30 l 0.2 g/l BSA

220

0.311

9,10

40 l 0.2 g/l BSA

210

0.351

11,12

50 l 0.2 g/l BSA

200

0.401

13,14

250 l unk, 1/20 dil

0.155

15,16

250 l unk, 1/6 dil

0.271

17,18

250 l unk, 1/2 dil

0.434

average
A595
blank

g / l
protein
(sample vol)

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AI-5

2.9. A solution of unknown protein concentration (UNK) was assayed by the Bradford protein assay,
using the microplate assay protocol. Below are the data obtained from the assay. Fill in the table, plot
the standard curve, and determine the protein concentration from the data.
sample volume
duplicates
BSA (0.024 g/l)
H2O
or UNK sample
0.0 l BSA
125 l
10.4 l BSA
114.6 l
20.8 l BSA
104.2 l
41.7 l BSA
83.3 l
62.5 l BSA
62.5 l
83.3 l BSA
41.7 l
105 l BSA
20 l
125 l BSA
0.0 l
1
125 l (1/3) dilution of UNK
125 l (1/3)2 dilution of UNK
125 l (1/3)3 dilution of UNK
125 l (1/3)4 dilution of UNK
125 l (1/3)5 dilution of UNK
125 l (1/3)6 dilution of UNK
125 l (1/3)7 dilution of UNK
125 l (1/3)8 dilution of UNK

Bradford
reagent
125 l

g / l BSA
(sample
volume)

A595
average

A595 average
minus blank

0.398
0.464
0.518
0.612
0.689
0.753
0.818
0.848
1.211
1.099
1.104
1.075
0.917
0.673
0.504
0.396

2.10. A new dye-binding protein assay is compared with the biuret protein assay. The new procedure
calls for mixing 1.5 ml of protein solution with 1.5 ml of reagent and reading the A630 after 3
minutes. To 0.5 ml of a BSA solution was added 1.5 ml of water. The assay was performed on 1.5 ml
of this diluted BSA solution, giving an A630 of 0.470. One-half ml of the original BSA solution was
mixed with 0.5 ml of water and 4.0 ml of biuret reagent. After 30 minutes the A540 of the solution
was 0.305. Assuming the same optical paths for both readings, proper use of blanks, both assays in
their linear ranges, etc., what is the relative sensitivity of the dye-binding and biuret assays?
Problems 2.11 2.14 are challenge problems.
2.11. It is possible to study tight binding of a small cofactor to an enzyme by mixing the two in solution
and then centrifuging to pellet the enzyme-cofactor complex. The experiment is normally performed
with excess cofactor. Unbound cofactor remains in solution after centrifugation and washed away. It
is assumed that any cofactor found with the enzyme pellet is bound.
This study was performed using 20.0 mg of an FMN binding enzyme with M.W. 200,000 and an
excess of FMN. The entire pellet of enzyme-FMN was dissolved in water to give a total volume of 15
ml. The absorbance of this solution at both 260 nm and 280 nm were A260 = 0.95; A280 = 1.33.
How many moles of FMN are bound per mole of enzyme in the pellet?
5

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If the enzyme bound one FMN per subunit, then what is the estimated protein subunit molecular
weight?

2.12. In (A) Spectrum of Protein or Nucleic Acid, are the separate spectra of aqueous solutions of known
concentrations of protein or nucleic acid; in (B) Spectrum of a Mixture, the spectrum is of a mixture
of protein and nucleic acid. Calculate the protein and nucleic acid concentrations in the mixture (B).
(A) Spectrum of Protein or Nucleic Acid

MCB120L

AI-7

(B) Spectrum of a Mixture (of protein and nucleic acid)

2.13. A new procedure (an assay) for the determination of inorganic phosphate is available. A bottle of
the assay reagent, Phosphate Reagent (a liquid), includes a brief method written on its side:
"Mix one part of reagent with two parts of water. Add one part of diluted reagent to one part of a
phosphate containing solution, heat at 100oC for ten minutes, cool to room temperature, and read at
660 nm. The reagent+phosphate chromophore is stable at room temperature; and has an absorption
coefficient, a = 1.24 x 105 (mg P/ ml)1 cm1 (P is elemental phosphorus --- atomic weight = 31)".
Write-up a protocol to assess the useful range of phosphate for the assay. Consider the following
points:
a) What range of molar potassium phosphate concentrations (in the cuvette) is used for the standard
curve? Explain and show calculations.
b) If one working stock solution of phosphate is made for the preparation of the standard curve, what
concentration would be chosen? Why?
c) Using the answer to b above, set up a protocol table for a standard curve.
d) How might the method be modified to increase its sensitivity so that the phosphate content of very
dilute solutions cab be determined accurately?
2.14. In Dr. Faircloughs lab -Bungarotoxin (-Btx, MW = 8000 protein) has been labeled with a dye:
carboxytetramethylrhodamine hydroxysuccinimide ester (Rh). The unreacted dye is removed with a
P-2 gel filtration column. Assume that this procedure removes all non-covalently associated dye
from the protein; only covalently attached dye remains and the protein is highly fluorescent.
An initial characterization of the labeled Btx derivative, Rh--Btx, involved recording a visible
absorption spectrum, Rh--Btx Spectrum, of a 1/50 dilution of the Rh--Btx solution. Also, using a
1.0 mg/ml unlabeled -Btx stock solution, a standard curve was prepared for a novel protein assay,

MCB120L

AI-8

read at 700 nm. The protocol for this assay is shown in Table I (next page) as well as the tabulation
of the A700 of the standards and several dilutions of the Rh--Btx unknown.
Rh--Btx Spectrum
0.8
0.7
0.6

Absorbance

0.5
0.4
0.3
0.2
0.0
0.0

400

450

500

550

Wavelength

600

650

700

(nm)

Table I
l Btx stock

l H2O

ml BCA

g -Btx

A700

0.0
4.0
8.0
12.0
16.0
20.0
20.0 (1/2 dilution Rh--Btx unknown)
20.0 (1/5 dilution Rh--Btx unknown)
20.0 (1/20 dilution Rh--Btx unknown)

20.0
16.0
12.0
8.0
4.0
0.0
0.0
0.0
0.0

1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0

0.0
4.0
8.0
12.0
16.0
20.0

0.100
0.130
0.160
0.190
0.220
0.250
0.310
0.176
0.109

From the data above, work-up the following points:

a) First, prepare a standard curve for the protein assay and get an equation for A700 vs. g -Btx.
b) Second, determine the protein concentration of the Rh--Btx derivative in mg/ml (show calculations).
c) Third, determine the molar -Btx concentration (in units such as M, mM, M, nM, pM, etc).
d) Fourth, determine the molar dye concentration in the Rh--Btx derivative assuming a molar
absorption coefficient, 550 = 80,000 M1 cm 1 (show calculations).
e) Fifth, in this preparation, calculate the average number of moles of dyes / moles of -Btx.
f) Sixth, how many l of the Rh--Btx are needed to add to 1.0 ml of water to have a solution with an
A550 = 1.0?
g) Lastly, what is the [protein] in this A550 = 1.0 solution (in M)?

MCB120L

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Practice Problems: Experiment 3

3.1 From the continuous assays performed with different enzyme concentrations shown below:
a) What are the initial velocities of the reactions for Curves AE?
b) Relative to Curve B, how much enzyme was used to obtain Curves D and E?
c) Draw the curves that would be obtained with 0.50X and 16X the amount of enzyme.
d) Fixed-time assays are those performed by stopping the reaction at a given time instead of
continuously monitoring the production of product. The reactions are stopped by denaturing the enzyme
(heat, acid, organic solvents, etc.), or by the addition of an inhibitor. The amount of product formed at
the time is then determined and a velocity calculated. If fixed-time assays are run with the amount of
enzyme shown in Curves A through E, what time interval are chosen for each enzyme concentration?
Why?

e) For routine fixed-time assays, which concentration of enzyme would be chosen? Why?
f) If Curve C was obtained by addition of 65 g of protein, what is the specific activity of the enzyme
preparation? What assumption is made in order to say that this is the specific activity of the enzyme?
g) Assume that Curve E was obtained with 12 picomoles of pure enzyme that has a molecular weight of
80,000 and two identical subunits, each with one catalytic site. Calculate the catalytic center activity and
molecular activity of the enzyme. Calculate the specific activity of the enzyme (IU/mg protein).

MCB120L

AI-10

h) Assume that Curve B was obtained as follows. A stock solution of enzyme was diluted 1/10 to
produce Dilution #1. Dilution #1 was then diluted 1/25 to produce Dilution #2. Dilution #2 was again
diluted 1/5 to produce Dilution #3. A 0.10 ml aliquot of Dilution 3 was used to produce Curve B. A 0.50
ml sample of Dilution 2 was found to contain 32 g of protein by the Coomassie Blue assay. Calculate
the specific activity of the enzyme using the same assumption as in part g above.
i) Calculate the total number of units of enzyme (total IU) contained in 7.0 ml of the stock enzyme
solution in part h above.
j) Suppose that Curve B was obtained with an enzyme with a Km = 40 M for the substrate and that the
substrate concentration was 160 M. Draw the initial rate portions of the curves that would be obtained
with 40 M and 10 M substrate.
3.2. Glyceraldehyde-3-phosphate dehydrogenase (G-3-P) catalyzes the following reaction:
G-3-P + Pi + NAD+ 1,3-diphosphoglycerate NADH + H+
To purify this enzyme, chicken liver is homogenized with isolation buffer, and the cellular debris is
removed by centrifugation. The protein concentration of the supernatant is determined to be 2.5 mg/ml.
G-3-P assays are performed on the supernatant fluid (0.1 ml of enzyme solution in a 3.0 ml final volume
reaction mixture containing appropriate concentrations of substrates and buffer). The results are:

a) Which dilution is the best for estimating the amount of enzyme present? Why?
b) What is the specific activity of the original extract? ( at 340 nm, NADH = 6220 M-1 cm-1)

10

MCB120L

AI-11

3.3. A new lactate dehydrogenase (LDH) is isolated from the muscle of a Torpedo fish. Fixed-time
assays are conducted for LDH in a 1 ml final reaction volume, with appropriate substrate concentrations,
and buffer. For the kinetic assays, 2 ml of a 1/10 dilution of the enzyme solution was prepared for a
working stock solution. The reactions are started by adding 30 l of enzyme working stock solution to
970 l reaction mix cocktail, and measured at 340 nm, for 20 seconds. The original enzyme solution had
a protein concentration of 0.063 mg/ml. The results are tabulated below and are used to determine the
turnover number for this Torpedo LDH (MW 38,400 Daltons) and to compare these results to beef
muscle LDH. ( at 340 nm, NADH = 6220 M-1 cm-1).
a) Calculate mg of enzyme in each sample and the number of IUs of enzyme assayed in each sample
(complete the table below, showing a sample calculation for each column).

Sample
1
2
3
4
5
6

working stock
solution of enzyme,
ml (dilution)
0.03 (undil.)
0.03
(1/2)
0.03
(1/3)
0.03
(1/6)
0.03
(1/9)
0.03 (1/12)

mg
enzyme

reaction mix
cocktail, ml

A340/20sec

0.970
0.970
0.970
0.970
0.970
0.970

0.0270
0.0135
0.0090
0.0045
0.0030
0.0022

mole P/min

b) Prepare a graph of IU vs mg of enzyme. Using this graph, calculate the specific activity of the
Torpedo LDH. (Hint: before proceeding with the IU calculations, make sure the rates are linear).
c) Now use the specific activity to calculate the turnover number for Torpedo LDH.
d) Is the Torpedo LDH faster or slower than bovine muscle LDH? Compare it to the bovine LDH:
140,000 Daltons with a turnover number of 7000 min1.
3.4. A mutant acetylcholine esterase is isolated from the Torpedo fish. The enzyme is assayed by
measuring the rate of production of choline in the following reaction:
H H +
CH COCCN(CH3)3
3
H H
O

+ H2O
Acetylcholine

H H +
HOCCN(CH3)3
H H

esterase

CH CO
3

O
Acetate

Choline

Acetylcholine

The rate of choline production also is assayed in the presence of a competitive inhibitor of the
esterase: 15 M compound I:
H

H H +
CH3CNCCN(CH3)3
H H
O
Compound I

11

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The results are in the table below:

Sample

acetylcholine
M

1
2
3
4
5

8.33
12.50
25.00
100.00
25.00

1/[sub]
M-1

Inhibitor
M

Vo
nM min-1

----------------15.00

9.52
13.33
22.22
44.44
13.33

1/Vo
nM-1 min

a) Prepare data for a Lineweaver-Burk plot (ie complete the table). Show units.
b) Draw the Lineweaver-Burk plot of the data, label the axes, and indicate the units.
c) Estimate Vmax and Km for the enzyme from the Lineweaver-Burk plot.
d) Estimate Kmapp from the Lineweaver-Burk plot using the definition of competitive inhibition.
e) Estimate the Ki for the inhibitor from the Lineweaver-Burk results.

12

MCB120L

AI-13

Practice Problems: Experiment 4

4.1. Formulate the equilibrium expression for the reaction below, in the direction written. What is meant
by the apparent equilibrium constant? Why is it pH dependent?
lactate + NAD+ pyruvate + NADH + H+
4.2. LDH activity can be assayed in either direction. What are the advantages and disadvantages of
assaying LDH in one direction or the other (ie +d(NADH)/dt or d(NADH)/dt )? What practical
differences between the assays need be considered?
4.3. To measure the concentration of NADH in a cell homogenate, what wavelength would be chosen
for maximum sensitivity (explain), or for optimizing both sensitivity and specificity (explain)?
4.4. What is the isoelectric point of a protein? Why is it important to know the isoelectric point of a
protein that is being purified by crystallization?
4.5. A hypothetical enzyme, unisilase, molecular weight 35,000 daltons, hydrolyzes its substrate, unisil,
producing the blue-colored compound nisil. The molar absorption coefficient of nisil at 595 nm is
1.24 x 104. The usual assay procedure is to follow the production of nisil at 595 nm. A typical
reaction mixture contains 50 mM phosphate buffer, pH 7.2; 2 mM unisil; and 0.1 ml enzyme, all in
a total volume of 2.0 ml; using cuvettes with a 1 cm path-length. The data in the table below were
collected in the course of purifying unisilase. Complete the table:

Volume of sample
Dilution for protein determination
Protein concentration in diluted sample
Total protein (mg)
Dilution for enzyme assay
A595
15 sec.
30 sec.
45 sec.
60 sec.
75 sec.
90 sec.
Total activity (I.U.)
Yield of enzyme activity
Specific activity (I.U./mg)
Fold purification
Turnover number

Crude
Extract
85 ml
1/200
67 g/0.2 ml

Pure Unisilase

1/40
0.090

1/300
0.023

0.170
0.245
0.325
0.375
0.415

0.050
0.075
0.101
0.127
0.153

6 ml
none
205 g/0.1 ml

100%
1

4.6. The enzyme catalase is to be purified from beef liver. Ammonium sulfate fractionation of the crude
homogenate is used as a first step in the purification scheme. The fractionation of catalase by
ammonium sulfate is not known, so a systematic examination of increasing ammonium sulfate
13

MCB120L

AI-14

concentrations was performed. After adding ammonium sulfate, each fraction was centrifuged and the
supernatants were assayed for catalase. The results are shown in the table below.
Tube
crude extract
1
2
3
4
5
6
7

% Saturation
(NH4)2SO4
---------20
30
40
50
60
70
80

[Protein],
mg/ml
19.3
19.4
16.3
14.0
11.7
5.0
3.2
2.1

Activity,
I.U. ml1
1230
1200
1191
1000
224
79
41
6.0

Spec. Activity,
I.U. mg1

% Recovery
of Activity

a) How is the purest enzyme using ammonium sulfate fractionation obtained? Provide an explanation.
Note: several answers to this question are possible.
b) What is the specific activity resulting from the selected purification procedure?
c) What is the fold purification that is expected?
4.7 Two different ammonium sulfate fractionation schemes for use as a first step in the purification of
an enzyme are shown below. Complete the table and answer: Which of the two fractionation procedures
would be the best to incorporate as the first step in a multi-step purification procedure for the enzyme?
Crude
Protein (mg/ml)
I.U./ml
Volume (ml)
Specific activity
Fold purification
Total I.U.
% yield of activity

20
100
50

Scheme I
0.5-0.8 saturated pellet
dissolved in buffer
5.0
300
12

Scheme II
0.6-0.7 saturated pellet
dissolved in buffer
5.0
1000
2.5

4.8. The electron transport catalyst, plastocyanin, from the unicellular cyanobacterium, Microcystis
aeruginosa, has an isoelectric point of 4.8, a pH optimum of 8.0, and is most stable to long term storage
at pH 7.0 (4oC). In the purification of this plastocyanin, anion exchange chromatography is examined.
Two columns are prepared, one at pH 8.0 and one at pH 5.0; the solution containing the plastocyaninis
loaded onto the columns and each uses a linear KCl gradient as eluant. The results appear in the
following two plots on the next page.
Which of these two procedures should be adopted, or should another column be examined at pH 7.0?

14

MCB120L

AI-15

2.5

1.0

pH 5 Data

1.5

0.6

B
B B

1.0

0.4

______

Enzyme Activity

0.8

A280

B 2.0

0.5

B BB
B

0.0
0

0.2

0.0
B B B BBB B B B BB

10
15
20
Fraction Number

25

30

2.5

1.0

pH 8 Data

1.5

0.6

1.0

0.5

0.4

0.0 B BBB B BBB B B B BBB B

4.9.

10
15
20
Fraction Number

Enzyme Activity

280

0.8

______

B 2.0

0.2

25

0.0

30

Four groups (A, B, C, & D) of MCB 120L students purified LDH from steak. A continuous
assay for LDH in a 1 ml final reaction volume was used, in cuvettes with a 1 cm path-length,
reactions started by adding 30 l enzyme solution. Below are the data for the peak LDH
fraction from the AMP-affinity column for each group. Also tabulated are the results of the
Bradford protien assay.
a) Given 340 = 6220 M1cm1 for NADH complete the chart.
b) Which preparation of LDH has the highest specific activity?
c) Which preparation has the most IUs of LDH?
15

MCB120L

AI-16

d) Which preparation has the most protein in it?

Group
Volume of pure enzyme
Enzyme dilution for assay
A340/min
I.U./assay
I.U./ml of pure enzyme
Total I.U.
Dilution for protein assay
Volume assayed for protein, ml
g protein in assay
mg/ml undiluted pure enzyme
Total protein, mg
Specific activity, I.U./mg

4.10.

A
5
1/500
0.040

B
3
1/800
0.074

C
2.5
1/400
0.059

D
4
1/1000
0.049

1/50
0.5
5.86

1/100
0.5
7.85

1/100
0.5
4.68

1/100
0.5
8.30

A small neuropeptide is newly isolated from the brain. Molecular biological study revealed that a
gene family of three genes codes for this peptide. The expressed peptides vary by one amino acid
at position 5:
Peptide I:
MAVHEICG
Peptide II:
MAVHKICG
Peptide III: MAVHLICG
Design an ion exchange chromatography protocol that would separate these three peptides given
buffers of your choice, DEAE-Sepharose, CM-Sepharose, and salt.
Table of pKa values
amino acid
E
H
C
K
M
A
L
G
I

-NH3+
9.67
9.17
10.78
8.95
9.21
9.69
9.60
9.60
9.68

-COOH

side chain

2.19
1.82
1.71
2.18
2.28
2.34
2.36
2.34
2.36

4.25
6.00
8.18
10.53
-----------

a) First calculate the net charge of each peptide at pH values pH 6.5, 7.5 and 8.5. Helpful hint: if
the pKa of an ionizable species is equal to or greater than 2 pH units from the pH, then the
species is assumed fully protonated or unprotonated.
b) Predict the order of elution using DEAE (anion-exchange) or CM (cation-exchange) at the
various pH values.
c) Which column and pH achieves the best (theoretical) separation?
16

MCB120L

AI-17

4.11 Malate dehydrogenase (MDH) coverts malate to oxaloacetate as shown in the reaction:

malate + NAD+ oxaloacetate + NADH

Keq = 6.2 x 10-6 = [oxaloacetate] [NADH] / [malate] [NAD+]
a) Enterprising MCB 120L students wish to study MDH from yeast and purify it using AMP-affinity
chromatography as was done for lactate dehydrogenase (LDH). Why is AMP-affinity column a
reasonable first hypothetical purification method?
b) There is no known transition state analog to elute MDH from the AMP-affinity column as was
available for LDH (ie the NAD+-pyruvate adduct). From the tables of molecules and their Km values
or Ki values for competitive inhibitors of MDH below, choose a hypothetical elution condition.
molecule
malate
NAD+
oxaloacetate
NADH

Km (mM)
0.014
0.31
0.023
0.030

molecule
ATP
ADP
AMP
cAMP
citrate
thyroxine

Ki (mM)
1.36
1.34
0.95
0.56
0.021
0.40

c) The AMP-affinity column did not bind MDH as hypothesized; a parallel control experiment resulted
in binding LDH. Propose an explanation for the lack of MDH binding to the AMP-affinity column.
d) From the tables above, choose a ligand and an eluant couple for affinity chromatography that
potentially can be used to purify MDH.
citrate

thyroxine

AMP

17

NAD+

MCB120L

AI-18

Practice Problems: Experiment 5/6

5.1. A PCR reaction is used to amplify a 750 bp (bp = base pairs) DNA fragment, starting with 2000
copies of double stranded template DNA. The mass of one mole of base pairs is 660 g.
a. After 35 PCR cycles, how many copies are theoretically expected?
b. How many g of product are theoretically expected?
c. But, only 14 g of PCR product was isolated. Assuming no losses in isolation, how efficient was the
PCR reaction?
d. To clone the 750 bp PCR product into a 2500 bp expression plasmid, 100 ng of restricted plasmid is
ligated with with a 5-fold molar excess of restricted PCR product. How many ng of PCR product is
needed?
5.2. A mass ruler (ie nucleic acid molecular weight standards) has the following characteristics:
Band Band
Band
length, bp mass, ng
A
2000
500
B
1000
250
C
400
100
D
100
25
a. A restriction digest shows two bands, X and Y. Xs length is the same as mass ruler band A and its
intensity is the same as band C. Ys length is the same as band B and its intensity is the same as band
C. Fill in the table below. It may help to draw a sketch of the gel.
Band Length Mass Mass/Length ratio
X
Y
What is the molar ratio of X/Y?
b. On another gel, band W is the same size as band D and as intense as band B. Band Z is the same size
as band B and as intense as band A.
Band Length Mass Mass/Length ratio
W
Z
What is the molar ratio of W/Z?
5.3 The cloning of genes that code for proteins from bacteria is simpler than from eukaryotes since
prokaryotic genes do not have introns and do not involve (extensive) RNA processing to express a
protein. The primary transcript of a prokaryotic gene is typically the mRNA that is translated directly (ie
without modification of the primary transcript). So, the cloning of bacterial genes by PCR is
accomplished by creating primers that recognize gene of interest, and using genomic DNA as the source.

18

MCB120L

AI-19

Below are the protein and gene sequences, respectively, for Malate Dehydrogenase (MDH) from the
cyanobacterium, Synechocystis PCC 6803.
MNILEYAPIA
QSVEEYDSKI
SPNAILIVVT
LVLGGHGDLM
ASSAAVMVES
AALHLSAEAV
atgaatattt
gctggcaatg
gttttgttgg
cagagcgtgg
ggctccgatg
gatttgttgg
tcccccaacg
tggaaagtaa
gctcggctca
ttagtgctgg
ggggttccca
cgtaacggtg
gcctcttccg
gccgccacct
cgtttggggt
gctgccctcc
gttagcgacg

CQSWQVTVVG
IGTNEYEATA
NPLDVMTYLA
LPLPRYCTVS
ILRNQSRILP
RLNIDVALAM

tggagtatgc
tggggcggac
acattgtgcc
aggaatacga
tggtggtaat
gcaaaaacgc
ccattttgat
ctggtttacc
aggccttcat
gcgggcacgg
ttaccgaatt
gggctgaaat
ctgcggtgat
accttgatgg
gtcgaggagt
atctttctgc
ggaagatatt

AGNVGRTLAQ
GSDVVVITAG
WKVTGLPSQR
GVPITELIPP
AATYLDGAYG
VSDG

tccgatcgcc
ccttgcccag
aggcttaccc
cagcaaaatc
taccgctggt
caacattgtg
tgtggtcacc
ttcccaacgg
tgcgatgaaa
agatttgatg
aatacccccc
tgccgcctta
ggtggagtcc
tgcctatgga
ggaagatatt
agaagcagtt
cgcgatggtt

RLVQQNVANV
LPRRPGMSRD
VMGMAGVLDS
QTIEELVERT
LKDIFLGVPC

tgtcagtcct
aggttagtgc
cagggcattg
attggcacca
ctaccccgca
gcccaggggg
aatcccctgg
gttatgggca
ttaggggcct
ctgcccttgc
caaaccattg
ctacaaacgg
attttacgca
ttgaaggaca
ctcgaagtgc
cgccttaata
agtgacgggt

VLLDIVPGLP
DLLGKNANIV
ARLKAFIAMK
RNGGAEIAAL
RLGCRGVEDI

ggcaggttac
agcaaaatgt
ccttggattt
atgaatacga
ggcccggcat
cccgggaagc
atgtaatgac
tggcgggggt
gtccttctga
cacgatactg
aagagttggt
gcacagccta
atcagtctag
ttttccttgg
aattaacccc
ttgatgtggc
aa

QGIALDLMAA
AQGAREALRY
LGACPSDINT
LQTGTAYYAP
LEVQLTPEEK

cgtggtcggc
cgccaacgta
gatggccgcc
ggccaccgcc
gagtcgggat
attgcgttat
ctatttggcc
gttggactcg
tatcaacacc
caccgtcagc
ggagcgtacc
ttatgcgccg
aattctcccc
agtgccctgc
tgaagaaaaa
gttggccatg

60
120
180
240
300
333
60
120
180
240
300
360
420
480
540
600
660
720
780
840
900
960
1002

Design both the forward (aka sense) primer and the reverse (aka anti-sense) primer to amplify the entire
MDH coding region by PCR. Make the primers at least 18 nucleotides long, and include the stop codon.
5.4 Most eukaryotic genes have introns, so the cloning of protein coding genes from eukaroyotes
typically involves creating a DNA copy of the mRNA since RNA processing removes the introns from
the primary RNA transcript. Poly(A+) RNA is isolated from the tissue of choice, then copied by the
enzyme, Reverse Transcriptase to produce DNA copies of (all) the mRNA present, which is called
cDNA. The gene of interest is copied from this population of cDNA by the usual PCR method. These
two steps are used so often that they are coupled together in a process called: RT-PCR (Reverse
Transcriptase PCR); the researcher supplies the specific primers and target mRNA.
Also, many other proteins include transit or signal peptide sequences at the N-terminus, and the proteins
are referred to as pre-proteins (eg pre-plastocyanin), which are later removed to produce the mature,
functioning, cellular protein. The transit and signal sequences are used in the cellular localization of the
protein. The transit sequences direct proteins to either the mitochondria or to chloroplasts after
translation is complete; signal sequences direct proteins during translation to the endoplasmic reticulum
(the beginning of the secretory pathway). The important point for the cloning: the transit and signal
sequences remain on the mRNA after RNA processing since these protein sequences are needed. If the
expression of the mature protein is the goal, then the primers are designed to begin copying the mRNA
at the N-terminus of the mature protein, and often, a methionine codon is added to provide a start codon
for the cloned protein.
Design both the forward (aka sense) primer and the reverse (aka anti-sense) primer to amplify the
plastocyanin coding for the mature protein region by PCR. Make each primer 18 nucleotides long.
19

MCB120L

AI-20

Below is the sequence information for the protein, the gene, and the mRNA of plastocyanin from
spinach (from the Expasy.org website: Swiss-Prot/trEMBL query).
Protein sequence:
Molecule processing
Transit peptide 1 69
Chain
70 168
10
20
30
40
50
60
MATVASSAAV AVPSFTGLKA SGSIKPTTAK IIPTTTAVPR LSVKASLKNV GAAVVATAAA
70
80
90
100
110
120
GLLAGNAMAV EVLLGGGDGS LAFLPGDFSV ASGEEIVFKN NAGFPHNVVF DEDEIPSGVD
130
140
150
160
AAKISMSEED LLNAPGETYK VTLTEKGTYK FYCSPHQGAG MVGKVTVN

Gene sequence:
gagcacaacc
taatccaatg
tcacctcatc
tctttggcaa
ttccattaca
ccttaaggcc
cgtcccaaga
cgcagccgcc
tgacggatca
attcaagaac
cggtgtcgac
aacttacaaa
gggtgctggt
ttatttatat
tagcacattt
taatccaaaa
agataaaatg

aatgaaaaac
ccactaaaaa
tatcctccat
ttgtcatttc
atggccaccg
tcaggatcca
ttgtctgtca
ggacttctag
ttggcattcc
aatgccggat
gccgcgaaga
gttaccctta
atggtgggaa
gactttgttg
gtgtagagct
ttggattata
ccaccattca

tagataatac
tcccacaaat
tttatcacct
ttaattgcat
tcgcttcctc
tcaagcctac
aggcttcctt
ccggaaacgc
ttccaggaga
tcccccacaa
tttcgatgtc
ctgagaaagg
aagtaactgt
tagagctatt
atttattcga
ccggtttaga
atctttatct

cctttattgg
gaaaaccaca
cttattaaat
tccacttaaa
cgctgccgta
caccgcaaaa
gaagaatgtc
catggccgtt
cttcagcgta
cgtagtgttt
cgaggaggat
aacttacaag
caactaatat
tatataaacg
aatttggttt
tttgaattga
gaaagaattc

cccacacatc
caaaaccatg
cccatcctcg
cccctccaaa
gccgtcccct
atcatcccaa
ggagccgccg
gaggtgttgc
gcctcgggcg
gacgaggacg
ttgctgaatg
ttctactgct
tttaatgaaa
atctcatcaa
agatttgatc
actacagtac

acaaatcctt
taaacagaca
ctctcagcac
atcctcctcc
cctttaccgg
ccaccaccgc
tggtagcaac
tcggaggggg
aggagatcgt
agattccctc
caccagggga
caccccacca
aattgaattt
ttggtacgtt
tcataaatca
tacactccta

60
120
180
240
300
360
420
480
540
600
660
720
780
840
900
960
1000

gccgtcccct
atcatcccaa
ggagccgccg
gaggtgttgc
gcctcgggcg
gacgaggacg
ttgctgaatg
ttctactgct

cctttaccgg
ccaccaccgc
tggtagcaac
tcggaggggg
aggagatcgt
agattccctc
caccagggga
caccccacca

ccttaaggcc
cgtcccaaga
cgcagccgcc
tgacggatca
attcaagaac
cggtgtcgac
aacttacaaa
gggtgctggt

60
120
180
240
300
360
420
480
507

cDNA sequence (derived from mRNA):

atggccaccg
tcaggatcca
ttgtctgtca
ggacttctag
ttggcattcc
aatgccggat
gccgcgaaga
gttaccctta
atggtgggaa

tcgcttcctc
tcaagcctac
aggcttcctt
ccggaaacgc
ttccaggaga
tcccccacaa
tttcgatgtc
ctgagaaagg
aagtaactgt

cgctgccgta
caccgcaaaa
gaagaatgtc
catggccgtt
cttcagcgta
cgtagtgttt
cgaggaggat
aacttacaag
caactaa

20

MCB120L

AI-21

Practice Problems: Experiment 7

7.1. The enzyme catalase contains approximately 116 residues of arginine, 204 of aspartic acid, 144 of
glutamic acid, 68 of histidine, 120 of lysine, and 84 of tyrosine per molecule. Make a rough estimate
of the net ionic charge of catalase at pH 7.5 and pH 9.5.
7.2. The lower reservoir buffer is about 250 ml of a TrisHCl buffer (0.05 M) pH 8.3. Gels are often run
for about 3 hours at 40 ma. A century ago, Michael Faraday discovered the relationship between the
amount of current passing from electrode to solution and the amount of chemical converted at that
electrode. He found that a transfer of 96,500 coulombs (now called a Faraday in his honor) of charge
corresponds to a release of 1 equivalent of reactant. An ampere is a current of 1 coulomb/second.
Remembering the reactions that take place at the anode, decide how much the pH will change in the
lower reservoir if a gel is run for 3 hours at 40 ma. The pKa of the amino group of Tris is 8.1. Do
you think you could reuse the buffer for a second run?
7.3. A solution contains the following three pure proteins:
Protein
A
B
C

Native MW (daltons)
100,000 (dimer, identical units)
80,000
50,000

pI
5.8
10
7.1

a) If this mixture were run on a Native-PAGE system identical to the conditions used in the lab (no
stacking gel), how many bands would be visible after Coomassie Blue staining? Why?
b) If a 10% SDS-PAGE system were run identical to the conditions used in lab, how many bands would
be visible after Coomassie Blue staining? Why?
c) If a 10% SDS-PAGE system were run similar to the conditions used in lab, with the exception that 2mercaptoethanol was omitted from the 2X SDS-sample buffer, then what might be the results after
staining with Coomassie Blue? Why?
7.4 Below is the reaction for the enzyme: theobromine demethylase (TD), which was isolated from the
muscle tissue of the Lesser Zimbu Monkey. Scientists at the UCD Genome Center have sequenced
the LZM genome and two isoforms of TD were found in the databanks.
TD
Theobromine + NADH + H+ + O2
NAD+ + H2O + CH2O + 3-Methylxanthine
Compute pI/MW (from the Expasy web site: protparam)
TD_A_ZIMBU (Q9BE24)
A chain (EC 1.1.1.27)
(TD-A: Theobromine Demethylase).
(Lesser Zimbu Monkey).
FT CHAIN 2 - 318 TD-A chain.
Molecular weight (average): 35600.48
Theoretical pI: 8.47

TD_B_ZIMBU (Q4R5B6)
B chain (EC 1.1.1.28)
(TD-B: Theobromine Demethylase).
(Lesser Zimbu Monkey)
FT CHAIN 2 - 320 TD-B chain.
Molecular weight (average): 36507.30
Theoretical pI: 5.76

Samples from the TD purification steps were analyzed by 6% Native-PAGE and histochemically
stained. Below is a copy of the histochemically stained native gel.
21

MCB120L

AI-22

a). Briefly explain the most likely composition of the three TD isozymes observed in the histochemical
stained bands (show any necessary work used to arrive at your conclusions).
b). In addition to the reagents PMS and NBT for the histochemical staining, what else is needed to
specifically identify TD?
7.5 The sea slug, Elysia chlorotica, eats algae, and captures and stores the algal chloroplasts in its own
cells. The captured chloroplasts continue to photosynthesize and fix carbon into glucose, which is
used by the sea slug. Former MCB 120L students chose to investigate two soluble cytoplasmic
dehydrogenases from the sea slug involved in glucose utilization paths: Glyceraldehyde-3-P
Dehydrogenase (GPDH) and Lactate Dehydrogenase (LDH).
triose-3-P + NAD+ 1,3-bis-P-glycerate + NADH + H+
lactate + NAD+
pyruvate + NADH + H+

GPDH
LDH

a) These enzymes have never been purified from the sea slug. Prior to running a Native-PAGE
analysis, briefly explain what could be done to make an educated estimation of the possible molecular
weights and isoelectric points of LDH and GPDH from sea slugs.
b) Briefly explain how to distinguish between LDH and GPDH by Native-PAGE when using
phenazinemethosulfate and nitroblue tetrazollum in the histochemical staining technique.
c) The Native-PAGE revealed which band is GPDH and LDH (only one isozyme for each), each band
is cut out of the gel, prepared for, and analyzed by, 10% SDS-PAGE. Only one band is observed for
each enzyme with Coomassie blue staining. Determine the subunit molecular weight for each enzyme.

lanes
2
3

5
top of gel
Lanes assignments
1. crude extract
2. 20% supernatant
3. 40% pellet
4. affinity column flow-through
5. affinity column combined
eluted fractions
dye-front

22

MCB120L

AI-23
PROTEIN STANDARDS
bovine serum albumin
ovalbumin
carbonic anhydrase
-lactoglobulin
lysozyme

MW (DALTONS)
66,000
45,000
29,000
18,400
14,300

RM
0.25
0.37
0.51
0.67
0.75

SAMPLES
GPDH
LDH

RM
0.41
0.45

7.6 An analysis of protein composition by SDS-PAGE using a mini-gel apparatus as is Experiment 5

(approximately 1 mm gel thickness) usually requires from 0.5 g to 1.0 g of a single purified
protein for sufficient visualization after staining with Coomassie Blue, whereas 10 g to 20 g of
protein in a crude extract is required in order to see the compliment of stained bands.
a) For a crude extract of 16 mg/ml, describe the preparation and loading of 10 g protein in a final
loading volume of 20 l using 2X SDS-sample buffer.
b) For a purified protein of 0.5 mg/ml, describe the preparation and loading of 1 g protein in a final
loading volume of 10 l using 2X SDS-sample buffer.

23

Practice Problems

AI-24

Practice Problems: Experiment 8

8.1. The optimum time for harvesting E. coli cultures is when the cells have grown to late log phase,
which is usually determined by measuring the A600 of a culture in a spectrophotometer. An A600 of 0.6
is approximately 5 x 108 cells/ml, which corresponds to late log phase.
a. A measure at A600 of an E. coli culture is 0.15. If the doubling time of E. coli is 1/2 hour, how much
longer will it take for the culture grow to get to late log phase? (Doubling time is the amount of time
it takes for one bacterium to divide into two.)
b. After 4 ml of an E. coli culture is transformed with a 4000 bp plasmid (2600 bp vector + 1400 bp
insert) in late log phase, the cells are harvested and 18 g of plasmid is isolated from the cells. What
is the average number of plasmids per E. coli? (~660 g/mol base pairs.)
c. The 18 g of plasmid from (b) above is suspended in 50 ul of TE to create a stock of plasmid. To
recover the insert from the plasmid, 20 l of this stock is digested with 30 l of restriction enzyme
mix (final volume is 50 l). After 6 hours, 10 l of the digest is removed and 2 l of loading dye is
added; the 12 l mixture is run on an agarose gel. Assuming digestion is complete, how many bands
and what band lengths should be seen? How many ng of DNA will be in each band?
8.2. The MCB 120L students are asked to test a new antisense (ie reverse) primer for BVR expression.
The vector is pASK75. The PCR products were cloned into the Xba I and Sal I sites.
The sequences from the Xba I site through the strep-tag of the clones made from the PCR products given
by the old primer, the new primer, and non-recombinant pASK75 are shown below.
pASK75:
Strep Tag
TCT AGA ATG [ 96 bp] TCG AGG TCG ACC TGC AGG C
Xba I
Sal I
Pst I

Ser Ala Trp Arg His Pro Gln Phe Gly Gly stopstop
AGC GCT TGG CGT CAC CCG CAG TTC GGT GGT TAA TAA GCT T
Hind III

old primer:
Strep Tag
Ser Ala Trp Arg His Pro Gln Phe Gly Gly stopstop
TCT AGA ATG [900 bp] GAA AAT GGT CGA CCT GCA GGC
AGC GCT TGG CGT CAC CCG CAG TTC GGT GGT TAA TAA GCT T
Xba I
Sal I
Pst I
Hind III

new primer:
Strep Tag
TCT AGA ATG [900 bp] GAA GCT TGT CGA CCT GCA GC
Xba I
Hind III Sal I Pst I

Ser Ala Trp Arg His Pro Gln Phe Gly Gly stopstop
AGC GCT TGG CGT CAC CCG CAG TTC GGT GGT TAA TAA GCT T
Hind III

24

Practice Problems

AI-25

To make sure the clones have the correct inserts, minipreps of the plasmid DNA are prepared from each
and digested with Pst I, or Xba I and Hind III (ie double digest, two enzymes). The digests are run on an
ethidium bromide stained agarose gel, shown in Figure 8.1.
Figure 8.1. Agarose gel

Figure 8.2. SDS polyacrylamide gel

a. The mass ruler is an equimolar mixture of DNA fragments. There are 85 ng in the 750 bp band.
How many ng are in the 3000 bp band?

____________ ng

25

Practice Problems

AI-26

b. The pASK75 PstI digest (band labeled A) is the same intensity as the 750 bp band in the mass ruler.
There is 100 l of the PstI digest; 4.0 ul of 6X loading dye is added to 20.0 l of the PstI digest, and
then 14.0 l of this mixture is loaded onto the gel.
How much pASK75 is in the original 100 l of digest? _______ ng
c. In the spaces below the agarose gel, to the left of Clone ID, identify which of the Xba/Hind digests
are from: pASK75, old, or new.
Crude extracts of induced E. coli cultures transformed with each of the three plasmids were loaded onto
an affinity column for BVR made by linking biliverdin to Sephadex beads. The eluted fractions were
pooled and loaded on the SDS gel shown in Figure 8.2. The gel has been stained with Coomassie
Blue.
d. In the spaces below the SDS gel, to the left of clone ID, identify each lane as pASK75, old or
new.
e. Briefly explain which clone(s) will be detected by a Western blot that recognizes the strep tag?
8.3. A gene of 1000 bp was isolated by PCR. The 5-end of both primers had EcoRI sites attached. Thus,
the PCR fragment is inserted into the EcoRI site of the MCS randomly in either direction (Figure 8.3:
orientation A or B). There is a PstI restriction enzyme site within the PCR fragment; the relative location
of the PstI site is shown below (Figure 8.3). Devise a restriction mapping experiment that definitively
distinguishes between orientation A and B. Assume the distances between the restriction enzyme sites of
the MCS region are essentially negligible when analyzed by agarose gel electrophoresis.
Figure 8.3. Insertion of a PCR fragment into a plasmid MCS
EcoRI
PstI

SalI

EcoRI

PstI

orientation
A

XbaI

MCS
EcoRI

plasmid
4000bp

plasmid
3000bp

PstI

EcoRI digest and ligation

SalI
XbaI

PCR fragment, 1000bp

EcoRI
EcoRI

PstI

500

EcoRI

orientation
B
PstI

plasmid
4000bp

1000

bp

EcoRI
PstI
SalI
XbaI

26

Practice Problems

AI-27

8.4. MCB 120L students, TAs, and instructors had their DNA analyzed for neurotransmitter gene
expression. One gene associated with intelligence, and named the Smart Gene, is expressed at high
levels in all MCB 120L students, TAs, and instructors (or so we thought). The Smart Gene DNA
sequence is known, and the ORF sequence does not contain any introns (only the sense strand is shown).
An antibody to the Smart Gene protein can be purchased commercially.
Smart Gene sequence: 66 nucleotides
ATGATCTGTCTAGATTTGCTACGCTCGAGCGGGTCGGAATTCTCGGTGGTTATGGATCCCAATTGA
(Met)

(XbaI)

(XhoI)

(EcoRI)

(BamHI)

(stop)

a) MCB 120L students want to isolate the Smart Gene by PCR and clone it into the pASK75 vector
(shown below). Fill in the restriction enzyme sequence chosen for the primers (more than one
combination is possible; and fill in the proper nucleotides of the Smart Gene sequence (only 10 are
needed); the CC are spacers.
Restriction Enzyme Site
sense primer:
5
(forward primer)

10 nucleotides of the Smart Gene

C

Restriction Enzyme Site

antisense primer: 5
(reverse primer)
recognition sequences

10 nucleotides of the Smart Gene

C

name

Recognition Sequence

BamHI
EcoRI
KpnI
HindIII
SalI
XbaI
XhoI

5-GGATCC-3
GAATTC
GGTACC
AAGCTT
GTCGAC
TCTAGA
CTCGAG

Restriction enzyme

b) Was the stop codon from the Smart Gene included in the antisense primer, why or why not?

27

Practice Problems

AI-28

c) Below is a restriction map of the smart gene isolated by PCR from one MCB 120L student and one of
the instructors (Instructor X, not to be named). What do the data reveal?

Lane assignments:

Student (1 3)

1. PCR fragment (uncut)

Lanes:

Instructor X (4 6)

2. XhoI digest

bp
100

3. EcoRI digest

80

4. PCR fragment (uncut)

60

5. XhoI digest
40

6. EcoRI digest

20

7. MW markers

d) Offer a conclusion and an explanation for the results (there is more than one plausible explanation).
pASK75 vector

XbaI (118)

EcoRI (210) KpnI (222)

BamHI (231)
XhoI (237)
SalI (243)

tetA
promoter

HindIII (291)

strep-tag

Ori

pASK75

SpeI (2492)

(3266)bp)
(3266

StyI (2370)

tet R

r
Amp R
amp

NsiI (2219)

PvuI (1379)

28