STUDENT NAME: Jeremy Patrick Le

GROUP: 5 Thursday

Experiment 1: Experiment Record

Fill in the blank areas and answer the questions as instructed text
should be entered electronically where possible. Calculations can be
typed within the document, or hand-written then scanned and inserted
as an image.
Graphs must be scanned and embedded in the document (please refer
to p. 14 of the lab manual).
Submit via Turnitin, following the instructions on the LMS site.

Aims:

To use a pipette and check the accuracy and reproducibility of the method

To measure absorbance spectra using the Perkin Elmer Lambda 2

spectrophotometer.

Materials and Methods:

As per BCMB20005 laboratory manual (Semester 1, 2015):

Pipetting accuracy exercises, p. 3941

UV/VIS Spectrophotometry, p. 4146

Results
Part B: Pipetting accuracy and reproducibility
Complete the following table with the data from Ex1 Table 1 from the laboratory
manual (p. 41), and calculate the mean and percentage error for each
measurement.
ER1 Table 1: Measurements of dH20 containing 5 g mL-1 methyl red volumes
to assess pipetting accuracy and reproducibility
Pipet
te
setti
ng
(L)

Reading 1
(mg)

Reading 2
(mg)

Reading 3
(mg)

Mean
(mg)

% Error*
(%)

A20
5

0.00

16

16

15

17

16

0.06

55

57

59

56

57.33

0.04

180

181

179

180

180

0.00

A200

Experiment 1 Experiment Record

STUDENT NAME: Jeremy Patrick Le

GROUP: 5 Thursday

A1000
245

244

247

247

246

0.004

750

751

752

751

751.33

0.002

*The difference between the mean and the expected value should be expressed as a percentage of
the expected value (e.g. if the mean = 48 mg and the expected value = 50 mg, the percentage
error is 2/50 100 = 4%

Experiment 1 Experiment Record

Part C: Absorbance spectrum of potassium chromate

Insert the spectra of the diluted potassium chromate solutions above the figure
caption below. Label the x- and y-axes and include units.
Hint: For assistance, refer to the general information section of the lab manual.

1/1

1/3

Fig. 1. Absorbance spectrum of potassium chromate diluted 1/10

and 1/30

Experiment 1 Experiment Record

Part D: Absorbance spectrum of DNA

Insert the spectrum of the diluted DNA solution above the figure caption below.
Label the x- and y-axes and include units.

Absorbance

Fig. 2. Absorbance spectrum of DNA diluted 1/20

Wavelength

Calculations for Part C & D:

1

From Fig. 1 and Fig. 2, identify the wavelength where the Absorbance value
(Ordinate value) is at a maximum (i.e. what is max?)
a. Potassium
chromate
max

0.39376 (10) & 0.14381

(30)

Experiment 1 Experiment Record

b. DNA
0.5889

1. Using the Absorbance values at the wavelengths identified in Q1, calculate

the concentration (g mL-1) of:
a. Part C tube 1: Potassium chromate in the original solution (i.e. before the 1/10
dilution).
Step 1: Calculate diluted concentration
0.39 = 268 %-1 cm-1 x 1 cm x c
c = 0.39 / (268 %-1 cm-1 x 1 cm)
c = 1.46 x 10-3 g 100mL-1 = 14.66 g mL-1
Step 2: Calculate original concentration
14.66 g mL-1 x 10 (dilution factor)
= 146.55 g mL-1
Potassium chromate has an extinction coefficient () of 268 % -1 cm-1 at 373 nm
(i.e. at 373 nm, the absorbance of potassium chromate will be 268 for a 1% (w/v)
solution (1 g/100 mL) measured in a 1 cm path length)
Hint: Use the Beer-Lambert equation to obtain the answer in % (w/v) and
convert it to g mL-1
b. Part C tube 2: Potassium chromate in the original solution (i.e. before the 1/30
dilution).
Step 1: Calculate diluted concentration
0.14 = 268 %-1 cm-1 x 1 cm x c
c = 0.14 / (268 %-1 cm-1 x 1 cm)
c = 5.37 x 10-4 100mL-1 = 5.37 g mL-1
Step 2: Calculate original concentration
5.37 g mL-1 x 30 (dilution factor)
= 160.98 g mL-1

Experiment 1 Experiment Record

c. DNA in the original solution (i.e. before the 1/20 dilution).

Step 1: Calculate diluted concentration
0.59 = 0.020 (g/mL)-1 cm-1 x 1 cm x c
c = 0.59 / (0.020 (g/mL)-1 cm-1 x 1 cm)
c = 29.45 g mL-1
Step 2: Calculate original concentration
29.45 g mL-1 x 20 (dilution factor)
= 588.9 g mL-1
Hint: Pure DNA has an extinction coefficient () of 0.020 (g/mL) -1 cm-1 at 260
nm, i.e. the absorbance of 50 g mL -1 pure DNA at 260 nm will be 1 AU
(Absorbance unit).
2. Was there any difference between the calculated concentration in Question
2a or 2b? If so, what does this difference suggest?
There is a difference of 14.43 g mL-1 between calculated concentrations for the
samples of dilution factor 10 and 30. This suggests that the dilution of
samples were inaccurate but because the absorbance values used to
calculate concentrations were incorrect (blunder), it is impossible to compare
it to the given concentration of potassium chromate (0.35 mg mL -1)

Discussion
Part B
Comment on the reproducibility and accuracy of your pipetting technique, and whether it
differed for the three pipettes tested.
The reproducibility and accuracy of pipetting technique was within approximately 5%
error throughout the three pipettes. Generally, pipettes became more accurate from A20
to A1000.
What steps could you take in future to improve the reproducibility and accuracy (if
necessary)?
Hint: Refer to Fig. 3.
The most obvious area that could be improved on is using the recommended immersion
depth to improve reproducibility and accuracy. Through practice, a rough idea on how
deep to immerse the tip can improve (e.g. 1-2mm for a small pipette).

Part C & D
Explain the differences in parameters and method of the wavelength scans
performed for the two different solutions (i.e. potassium chromate versus DNA).
The parameters used for the wavelength scans performed for potassium
chromate and DNA were almost identical except for the range of wavelengths
used. A visible light spectrophotometry was performed for potassium chromate
from 300 nm - 500 nm. DNA was scanned using UV spectrophotometry and
parameters were set lower from 200 nm - 350 nm.

Comment on the accuracy of your pipetting technique in preparing your

potassium chromate solutions.
Due to the difference in values calculated between the two solutions (14.43 ug
mL-1), it is assumed that the accuracy in preparing the solutions needed to be
improved to minimise discrepancies because they were prepared from the same
given solution.

Conclusion
Week 1's practical served as an introduction to techniques of molecular sciences
and laboratory work. Pipettes, scales, spectrophotometers, centrifuges were all
demonstrated during the practical followed by hands on practical work in diluting
solutions with pipettes and analysing them using spectrophotometry. Not
everything went according to plan, the values for Part C are wrong because the
cuvette was placed the wrong way in the spectrophotometer. However, mistakes
such as this as well as self-feedback from analysing accuracy of results will
become a learning experience and can only be improved upon.