Lab - Biological Sample Analysis Flashcards
Be able to determine the concentration of DNA (haemoglobin/blood glucose) in an “unknown” sample.
- The first step is to prepare working standards of DNA;
A. To do this you will measure out a series of different concentrations Of DNA
B. Label seven Eppendorf microtubes 1 to 7 and place in a microtube rack.
C. Using the appropriate size of automatic pipette, add to each tube the amounts of 50
micrograms per millilitre (50gg/ml) standard DNA solution needed to give the DNA concentrations indicated in the table.
D. Change pipette tip and/or pipette, then add the appropriate amounts of buffer to make
the total 1000gl (or Iml).
E. Cap each tube and mix.
- You now need to set up the spectrophotometer
A. Switch on the spectrophotometer and adjust the wavelength to 260nm, if necessary.
B. Empty the contents Of your blank (Opg/ml) Eppendorf into the cuvette.
C. Place the cuvette, IN THE CORRECT ORIENTATION (clear/smooth window sides in the light path), in the spectrophotometer. Always handle the frosted surfaces only. Close the spectrophotometer lid.
D. Press the “Set Reference Button” and the analyser should read 0.0 after a few seconds.
E. Empty your blank solution back into the Eppendorf as fully as possible, then rinse the cuvette with a small amount of distilled water. Ensure you empty the cuvette as fully as
possible after rinsing and ensure you do not get the outside of the cuvette wet.
F. Place the cuvette containing the first standard solution (5gg/ml) into the
spectrophotometer and record the absorbance (A260) in the table below.
Tube number
G. Repeat the process using standards of increasing concentration
- Determine the DNA concentration of the two “unknown” DNA preparations
provided.
Measure the absorbance Of the two “unknown” samples Of DNA and record the
absorbance
- You now should plot your calibration curve and determine the concentration of DNA in samples A and B..
The results need to be platted as a graph of A260 against DNA concentration to produce a calibration curve using the graph paper
Please ensure the following when constructing your graph:
i. The graph should be of reasonable size, with appropriate space for labelling the axes.
ii. The variable being measured (ie A260) should be plotted on the upright, y-axis.
iii. Both axes should be labelled clearly with units shown.
iv. The graph should have a suitable legend or title.
v. The points on the graph should be clear and, in this case, a line of best fit should be drawn
What is Spectrophotometry / Colorimetry and its function?
A type of analysis that is used for measurement of haemoglobin,
glucose, lactate or protein concentrations in the blood
A spectrophotometer or colorimeter makes use of the transmission of light through a solution to determine the concentration of a solute within the solution.
A spectrophotometer differs from a colorimeter in the manner in which light is separated into its component wavelengths.
A spectrophotometer uses a prism to separate light and a
colorimeter uses filters.
Both are based on a simple design of passing light of a known wavelength through a
sample and measuring the amount of light energy that is transmitted.
This is accomplished by placing a photocell on the other side of the sample. All molecules absorb radiant energy at one wavelength or another.
Those that absorb energy within the visible spectrum are known as pigments
How is Spectrophotometer set up?
The design of the single beam spectrophotometer involves a light source, a prism, a
sample holder and a photocell.
Connected to each are the appropriate electrical or mechanical systems to control the illuminating intensity, the wavelength, and for
conversion of energy received at the photocell into a voltage fluctuation.
The voltage fluctuation is then displayed on a metre scale, is displayed digitally, or is recorded via connection to a computer for later investigation.
What is required to work out an unknown samples concentration?
To use a Spectrophotometer it is necessary to establish a known series of dilutions
containing known quantities of a solute. One of these will contain no solute and is known
as the blank. It is used to adjust the instrument to read 100% transmittance or 0%
absorbance. The blank sample (containing no solute or dye) is inserted, the curtain
opened and the metre readjusted to read 100% transmittance. All Other measures are then
made by merely inserting the samples into the light path and measuring the %
transmittance. Most spectrophotometers have a built in means of direct conversion of this
reading to absorbance.
After recording the absorbance for a series of standards, a plot is made of the absorbance
value (y-axis) against the concentration (x-axis).
How do you use a pipette?
- Read and adjust the volume
- Put on a tip by pressing plunge to first position and lifting back up slowly
- Place tip in solution and push down to first position and back up slowly
- Place in Eppendorf microtubule and push down to first position, then second position and release cap back up slowly
