Experiment 1

A 60mM solution of unknown base 3 was used and its absorbance was measured using a
spectrophotometer, from 310nm to 230nm, according to the instructions on page 25 of the
MBLG2071 Laboratory Manual. Prior to this, a complete spectrum was obtained of the unknown
solution, using a cuvette full of TE (Tris-EDTA, the buffer and solvent used in the base solution) to
correct the baselines. A graph of the spectrum was obtained (Figure 1).
From this, the peak wavelength (max) and absorbance at the peak wavelength (Amax) were obtained
from the Peak option on the spectrophotometer (Table 1).
Since the peak was identified at 266nm, the absorbance was measured every 10nm between 310nm
and 280nm and 250nm to 230nm, and every 5nm between 275nm and 255nm, in order to get an
accurate value at the peak, and TE was used to blank the spectrophotometer at each reading (Table
2).

Figure 1: Absorption spectrum of a 60mM solution of unknown base 3, between 310 and 230nm, the baseline having been
corrected with Tris-EDTA.
Table 1: Details of the peak identified on the full spectrum in Figure 1.

max (nm)
266.0

Amax
0.344

Table 2: Absorption of the 60mM solution of unknown base 3 between 230nm and 310nm, blanked with TE.

max (nm)
310
300
290
280
275
270
265
260

A
0.000
0.000
0.018
0.175
0.268
0.332
0.343
0.305

255
250
240
230

0.241
0.162
-0.021
-0.299

From the absorbance values obtained, a graph was plotted using Excel (Figure 2) in order to compare
to the model graphs in the Laboratory Manual.
0.4
0.3

Absorbance

0.2
0.1
0
230

240

250

260

270

280

290

300

310

-0.1
-0.2
-0.3

Wavelength (nm)

Figure 2: Graph of measured absorbance values of a 60mM solution of unknown base 3.

The negative values at 240nm and 230nm (Table 2) are very unusual and indicate an error. The
entire spectrum (Figure 2 and Figure 1) was therefore not used to identify the base, and instead the
peak values (Table 1) were used.

We know that = = , so =

0.344
1 60103

= 5.731

Therefore, we have determined for our unknown base that max=266nm, max=5.73Mcm-1. This
corresponds mostly closely to the spectral properties of cytosine (max=265nm, max=5.8Mcm-1, from
the laboratory manual). Therefore, unidentified base 3 is cytosine.
We theorised that the spectrophotometer was defective below 250nm, since the lower end of the
graph does not match the spectrum for cytosine (from the laboratory manual), or that there could
have been some other type of contamination of the solution.

Experiment 2
The unknown solution 2 was selected, which was indicated to have a concentration of 0.75 to
1.5mg/mL. From this range, it was determined which dilution of this stock solution would give an
absorbance of 0.5 (the absorbance at which the spectrophotometer is most reliable) for the lower,
middle (1.125mg/mL) and upper limits of the concentration range:
50g/mL of DNA gives an absorbance of 1 (laboratory manual), so 25g/mL gives an absorbance of
0.5, which is therefore the concentration we want.

750
25

= 30, so the stock solution is 30 times more concentrated than we need it to be, so we want a

dilution factor of 30.

1125
25

= 45 dilution factor.

1500
25

= 60 dilution factor.

A 5mL solution was to be prepared for each of these dilutions in order to then have more than
sufficient solution for the 3mL cuvettes (Table 3).
To create the dilutions, a certain volume of the DNA solution (V(DNA)) was diluted in a certain
volume of TE (V(TE), the solvent which is used in the DNA solutions), for which the volumes were
calculated by:
5

VDNA = and VTE = 5mL VDNA. For example, for test tube 1, VDNA =

5
=0.167mL,
30

and VTE

= 5mL 0.167mL = 4.833mL.

The 5mL diluted solutions were thus created in 10mL test tubes, then the solutions mixed, then 3mL
of these solutions pipetted into 3mL quartz cuvettes. Using the stock solution of TE to blank the
spectrophotometer, the absorbance of each solution was determined at 260nm, then 280nm.
From this, the A260:A280 ratio was calculated by dividing the A260 by the A280, and the concentration of
the diluted solution (Cdil) was calculated by:
50g/mL has an absorbance of 1.0, therefore an absorbance of A corresponds to a concentration of
Ax50g/mL. For example, Cdil(test tube 1) = 0.680 x 50 g/mL = 34 g/mL.
The original concentration of the solution before dilution is calculated by:
Catual=dilution factor x Cdil, for example Cactual (test tube 1) = 34 g/mL x 30 = 1020 g/mL = 1.020
mg/mL.
The concentration of the original solution was therefore calculated for each dilution, giving 3
replicates from which the average was determined (Table 3).
Table 3: calculated volumes of stock solutions required to make each diluted solution, absorbance values and the calculated
A260:A280 ratio and concentration.

Test
tube
1
2
3

V(TE)
(mL)
4.83
3
4.88
9
4.91
7

V(DNA
) (mL)
0.167

Dilution
factor
30

Adil(260nm)

Adil(280nm)

0.680

0.111

45

0.083

60
Average

Cdil(g/mL)

Cactual(mg/mL)

0.371

A260/
A280
1.833

34.00

1.020

0.451

0.245

1.841

22.55

1.015

0.321

0.173

1.855

16.05

0.963

1.843

0.999

The original concentration of unknown solution 2 was in fact 1mg/mL, therefore our dilution method
determined the concentration quite accurately.

The A260:A280 ratio indicates that there is slight RNA contamination in the sample, since the ratio is
higher than 1.8 (the value for pure DNA) and RNA has a higher value of A260 (its peak wavelength as
well) due to the fact that it is most commonly single-stranded, and therefore there are less
interactions between strands which prevent the nitrogenous bases from vibrating as much upon
reception of UV radiation (and therefore absorbing as much UV radiation). This causes an upward
shift of the ratio.

Discussion
A A260:A280 ratio below 1.8 indicates protein contamination, since protein has a peak wavelength of
280nm, therefore A280 is higher than A260, and thus this shifts the ratio downward.
This ratio does not depend on the concentration of the solution, although very low concentrations
can cause the absorbance values, and thus the ratio, to be unreliable. This is because this ratio is an
intrinsic property of DNA.
This method cannot reliably be used to distinguish RNA from DNA since it is a semi-quantitative only,
and only gives an idea of how contaminated the sample is, although a ratio of 2.0 is often regarded
to be pure RNA. A ratio of 1.8 could also be caused by a mixture of RNA and protein instead of pure
DNA. Therefore, while RNA and DNA can more or less be semi-quantitively distinguished by this
method, it is not reliable enough.
The size of the nucleic acid cannot be determined by this method, since the size does not affect the
concentration, and this method only allows the concentration of the DNA to be measured. The size
of the DNA could be determined by gel electrophoresis, for example.