NOTES ON DNA INTERCALATION / PROFLAVINE FLUORESCENCE LAB
Donald J. Wink, [email protected]
October 2014
AMOUNTS TO BE USED IN FALL, 2014
Proflavine working solution: 3.0 × 10-6 M (3.0 M)
DNA working solution: 3.1 × 10-5 M (31. M) in base pairs (20 mg / L)
Salt solutions: 0.2 M NaBr, NaCl, KI, NaI, KCl
UPDATE TO PROCEDURE
Students should set up their solutions in Part 3 so they are about 1-1.5 M in proflavine and
within the range 3-15 M in DNA. The concentrations mentioned in the procedure are too high.
The solutions given, 3 M proflavine and 31 M DNA, are appropriate.
We have also obtained adjustable 10-100 L micropipets for the students to use. This will be
considerably easier than with Mohr pipets and should give more accurate results. These are
delicate and should be handled carefully. Note that in some cases students will need to make
multiple measurements in order to reach a desired volumes (e.g. 5 × 100 L to make 500 L).
When using the UV lamps, set the lamp to give shortwave UV light. This illuminates a different
excitation than blue light but also gives fluorescence from the same state.
1. Note on the experimental setup
This lab relies on illumination of a sample of proflavine, a dye that fluoresces (if it is not
quenched!) in the green with a peak at about 514 nm. We can excite the fluorescence with
shortwave UV or with blue light. We use UV lamps with special observation boxes and a bright
white light with a blue filter for illumination. Note that there are slight differences in the filters,
so there may be results that vary from lamp to lamp. But if students use the same lamp for each
measurement this will not be a problem.
2. Using spectrometer in fluorescence
mode
Physical arrangement of the spectrometer,
lamp, and cuvette. The spectrometer should
be hooked up to the computer for use. The
computer should be adjacent to a lab bench.
The spectrometer and illumination lamp
will be on the bench, as shown at right.
In order to get consistent results, it is
important to position the illumination light
and the spectrometer in the same places for
each measurement. We have marked off a
spot on the bench with tape. The
spectrometer should be positioned inside the tape. The lamp should
be placed so that the light is about 1 inch above the top of the
cuvette with the cuvette in the center of the illumination, as shown
at right. The lamp is not moved, but the spectrometer (with the
cuvette) can be moved in and out from under the lamp in order to
change cuvettes.
Starting and using the software. The experiment uses the
OceanView software from Ocean Optics. Upon starting the
software, you may get an error indicating that there is a missing
driver. You can click past this. The software then comes up with a
“Run a Wizard” option. Select that and then “Spectroscopy” and
“Quick View Fluorescence.”
You are given a parameter window. Set up the following
parameters:
Acquisition time: 2000 ms
Scans to average: 5
Boxcar width: 3
Other parameters are left in default mode. In particular, the lamp should not be “Enabled.”
Then, click “Next.” The system will then go into its background collection mode. Insert a blank
with approximately 1.0 mL of phosphate buffer for this. It takes ca. 12 seconds to collect and
process a spectrum. When the system is consistent, click the black light bulb icon and wait until
the background is displayed. The “background” should be collected with the illumination lamp in
position and turned on. Some scatter may occur that shows some of the blue illumination light in
the window. This is not a problem, as we observe at different wavelengths. Also note that,
depending on the position of the spectrometer in the room, there may be some of other lines due
to the mercury emission of fluorescent lamps. Again, if they are consistent with positioning, etc,
these are not a problem.
The values of the fluorescence will vary from spectrometer to spectrometer, in part because there
are different filters in use. But the important parameter is relative fluorescence, and the formula
given in the manual takes that into account.
Obtaining proflavine emission spectra. Place a cuvette containing an experimental solution in
the spectrometer. When positioned under the illumination lamp this should show a fluorescence
centered at 514 nm. If needed, rescale the window with the “up/down arrow” icon. You can read
the intensity of the fluorescence by clicking on the peak, as shown below (note that in this case
there is an inconsequential ‘negative’ peak in the blue, due to slight differences in some
backgrounds scatter of the illumination light). The counts are given below the graph window and
should be recorded. Some bleaching of the fluorescence may occur, so it is best to get the
intensity within the first 30 seconds or so.
3. Procedure guide
Part 2: Concentration dependence of fluorescence
This is a simple experiment that introduces students to the use of the spectrometers as
fluorescence instruments. The comparison with Beer’s law is to simply get students to realize
that fluorescence is directly proportional to concentration.
Part 3: Testing for quenching with metal salts.
Students need to carry out a serial dilution while keeping the concentration of proflavine
constant. This may take some explanation, since they will be tempted to try to make very dilute
solutions (1 × 10-6 M!) directly from 0.20 M stock solutions. It is a good opportunity for them to
practice their micropipetting skills. A sample layout of one of the well plates is given on the last
page, along with a picture of sample results. Note that NaCl does nothing, NaBr quenches a bit in
the first solution, and NaI completely quenches the 0.10 M and 0.010 M solutions. KCl also does
nothing but KI behaves like NaI.
Part 4: DNA fluorescence experiments.
As noted earlier, students should aim for 1-1.5 M proflavine
in each cuvette, but the value should be consistent to the
nearest 0.1 M. The values for DNA will range from 3-15 M
in DNA. They should gently mix the solutions and, if there is
time, inspect them in the UV box. Sample results are shown at
right (as with the other photos, this is with very low light!).
Layout of a typical tissue culture plate with serial dilution to study
quenching by sodium halide salts
A
B
C
D
E
1
2
3
1 × 10‐1 M NaCl 0.50 mL proflavine 0.50 mL 0.20 M NaCl 1 × 10‐1 M NaBr 0.50 mL proflavine 0.50 mL 0.20 M NaBr 1 × 10‐1 M NaI 0.50 mL proflavine 0.50 mL 0.20 M NaI 1 × 10‐3 M NaCl
0.10 mL “B1” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐4 M NaCl
0.10 mL “C1” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐5 M NaCl 0.10 mL “D1” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐6 M NaCl
0.10 mL “E1” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐2 M NaBr 0.10 mL “B2” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐3 M NaBr
0.10 mL “B2” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐4 M NaBr
0.10 mL “C2” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐5 M NaBr 0.10 mL “D2” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐6 M NaBr
0.10 mL “E2” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐2 M NaI 0.10 mL “A3” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐3 M NaI
0.10 mL “B3” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐4 M NaI
0.10 mL “C3” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐5 M NaI 0.10 mL “D3” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐6 M NaI
0.10 mL “E3” 0.45 mL proflavine 0.45 mL buffer Layout of a typical tissue culture plate with serial dilution to study
quenching by potassiumhalide salts
A
B
C
D
E
1
2
1 × 10‐1 M KCl 0.50 mL proflavine 0.50 mL 0.20 M KCl 1 × 10‐1 M KI 0.50 mL proflavine 0.50 mL 0.20 M KI F
1 × 10‐2 M NaCl 0.10 mL “A1” 0.45 mL proflavine 0.45 mL buffer F
1 × 10‐2 M KCl 0.10 mL “A1” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐3 M KCl
0.10 mL “B1” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐4 M KCl
0.10 mL “C1” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐5 M KCl 0.10 mL “D1” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐6 M KCl
0.10 mL “E1” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐2 M KI 0.10 mL “A2” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐3 M KI
0.10 mL “B2” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐4 M KI
0.10 mL “C2” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐5 M KI 0.10 mL “D2” 0.45 mL proflavine 0.45 mL buffer 1 × 10‐6 M KI
0.10 mL “E2” 0.45 mL proflavine 0.45 mL buffer Photo of NaCl, NaBr, and NaI quenching
experiment
Photo of KCl and KI quenching experiment