INTRODUCTION

Although some modern bioscience laboratories may require very specialized skills of laboratory workers most labs require workers who know certain basic lab skills. These skills include the ability to pipet, make solutions and dilutions, perform spectrophotometric analyses and to execute protocols which require the use of sterile techniques.

The transferring or measuring of fluids using a pipet is probably the most commonly performed operation in the lab. If you do not know how to pipet properly, your experiments will likely fail miserably or be irreproducible. Additionally, improper handling of glass pipets can lead to serious injury.

Proper preparation of solutions is another critical aspect in producing successful experiments. Not only must one know how to determine the correct quantities of solute and solvent to produce a required solution concentration but one must know how to use a balance and often a pH meter. Solution concentrations are expressed in several different ways and the laboratory worker should be familiar with all of them. Some of the most common ones in the bioscience laboratory include molarity, %, normality, and mg/ml. Other somewhat less commonly seen units of concentration include parts per million (PPM), molality, and mg%.

Absorbance of light (ultraviolet to visible to IR) by solutes in solution is used in a number of ways in bioscience laboratories. In this exercise absorbance of light will be used to show an absorbance (and transmission) spectrum for a solute and to demonstrate the relationship between absorbance (and transmission) of a solution and its concentration. In the next exercise spectrophotometry will be used to assay for the rate of an enzymatic reaction.

Absorbance Spectra

The absorbance spectrum, which is a graph of the absorbance of a solute as a function of wavelength, is unique for each solute and can be used to identify particular solutes in solutions. The absorbance spectrum is a result of the electronic, vibrational and rotational energy levels of the solute molecules. For example, chlorophyll in solution looks green because it absorbs quanta of light equivalent to the energy levels of blue and red light but not green light. Chlorophyll a has absorbance maxima approximately at wavelengths 450 nm and at 670 nm whereas nucleic acids have an absorbance maximum at 258 nm in the ultraviolet region. See Figure 1 and Figure 2 for representations of these absorbance spectra.

Visible wavelengths are roughly from 390 nm to 780 nm with the following rather arbitrary subdivisions:

violet 390 - 430 nm

blue 430 - 470 nm

blue-green 470 - 500 nm

green 500 - 530 nm

yellow green 530 - 560 nm

yellow 560 - 590 nm

orange 590 - 620 nm

red 620 - 780 nm

Beer-Lambert Law

The absorbance of a solute is directly related to the concentration of the solute and also to the length of the light path through the solution. The wavelength chosen to establish this relationship is usually the wavelength of the absorbance maximum for the solute and one which other components (solute or solvent) in the solution do not absorb well.

The Beer-Lambert law states:

Absorbance (A)= ecl; where

A (sometimes called Optical Density or OD if the light path length is 1cm) is dimensionless

e is the molar extinction coefficient in cm^-1 M^-1

c is the concentration in moles/liter (M) and

l is the length of the light path through a solution in cm

The law is based on the following: When a solvent in solution is interposed in a light beam the intensity of light transmitted through that solvent is I_o. When a solution consisting of the solvent and a solute is interposed the intensity of light is I. The fraction of light transmitted is

I/Io = Transmission (T). Since I and Io have the same units,

T is dimensionless. T has a range of values from 0 to 1.

The Percent Transmission is T(100) = Transmittance

(unfortunately spectrophotometer manufacturers call this percent

transmittance - oh well).

It has been established that T is a negative exponential function of concentration (Beer's law - see Figure 3) and of the length of the light path through the solution (Lambert's Law - see Figure 4). Thus,

T = 10-ecl and

Log 10T = -ecl and

Log 101/T = ecl and

Log 101/T is known as Absorbance (A). Thus

A = ecl; the Beer-Lambert law.

Absorbance is directly proportional to concentration and light path length and is a constant for a solute at a particular wavelength.

See Figure 5 for a graphical representation of A = ecl.

Principles of the spectrophotometer

A spectrophotometer consists of (1) a light source; (2) an adjustable monochromator (either a grating or a prism) for dispersing light into its constituent wavelengths; (3) a sample holder; (4) a shutter; (5) a phototube to detect the transmitted light; (6) an amplifier; and (7) a meter or digital display that usually can be read both in Transmittance (% T) and in Absorbance (A) units. After selection of the appropriate wavelength, a "blank" solution, containing all constituents except the solute to be measured is placed in the light path. The meter is set to read zero A and 100% T. The sample containing the solute in solution is then placed in the light path and its A or % T is read directly.

Spectrophotometers tend to be relatively inaccurate at high concentrations of solute. There is an optimum range of Absorbance, from 0.1 to 0.7, that gives the best accuracy. If the initial concentration of a solution gives an A which is greater than 0.7, the solution should be diluted. Since A and c are directly related, often the initial A above 0.7 can give an estimate of the dilution needed. For example, if the initial A = 1.5, a 1/3 dilution should give an A of approximately 0.5.

Beer's Law may not be followed over all concentrations. This may happen if association of the absorbing molecules occurs at high concentrations resulting in a new compound with a different absorbance spectrum. Also, at high concentrations solute molecules do not absorb light independently. One solute molecule may "shadow" another and the absorbance will be lower than predicted. Changes in temperature and pH may also affect the absorbance spectrum of a compound. Deviations from Beer's Law also occur if the light is not monochromatic--that is,

the band of wavelengths passing through the sample is not very narrow. More expensive instruments with intense light sources and highly sensitive photocells can utilize narrower wavebands.

Because most biological molecules are electrically charged they can move in an electric field. Electrophoresis is the movement of molecules in solution under the influence of an electric field. Electrophoresis is an extremely useful method for separating biological molecules to determine such properties as molecular weights, to distinguish molecules by their net charge or shape, to detect changes from charged to uncharged residues or to separate different molecular complexes quantitatively. Electrophoresis uses the rate of movement of a molecule in an electric field to separate different macromolecules. The rate of migration is dependent on the size and shape of the molecule, the charge of the molecule, the applied current and the resistance of the medium it is moving in.

The equation,

E q = f v

states that the electrical force E q is countered by the viscous drag, f v, where f is the frictional coefficient, v is the velocity, E is the electric field and q is the charge of the molecule. Note that the velocity is proportional to the voltage and not electrical current. There are further details of electrophoresis such as electrolytes in the solvent or supporting medium that complicate exactly how a molecule will behave but that will not be discussed in this course.

The most common type of electrophoresis used in biochemistry and molecular biology is called zone electrophoresis. In this type of electrophoresis, a solution containing the biological molecules is placed in a solvent that is supported by a chemically inert medium such as a gel or paper and the molecules migrate on or through the supporting medium. Using non-adsorbing gels such as polyacrylamide or agarose as the supporting medium can aid in separation of the molecules by acting as a molecular-sieve.

Power supplies used in electrophoresis can be set to either maintain constant voltage, constant current or constant power. You will often see conditions for an electrophoresis experiment described in terms of volts per centimeter. But most times the voltage given is the voltage at the power supply and not the voltage across the gel divided by the length of the gel. Since the voltage across the gel is decreased by the resistance due to the electrical wire leads and solution medium this is a only an approximation. Remember that Ohm’s law states that current I is equal to V/R where R is the total resistance of the circuit. In electrophoresis this means that a current set at the power supply, I, equals the voltage at the power supply,V , divided by the sum of the resistances, R, since the resistances are in series. What follows is that the voltage across the gel V_g = I R_g where R_g is the resistance of the gel and any overlying buffer. Therefore to reproduce the same velocity of the molecules in the gel,V_g and not V must be maintained. This is done by setting the current I constant and not V, the voltage at the power supply. So the voltage across the gel V_g is the force that moves the molecules through the gel and constant voltage is sustained by maintaining constant current through the circuit. Though maintaining consistent and reproducible conditions is beneficial to reproduce results in electrophoresis one must also keep in mind that that there is an upper limit to the voltage that can be applied to a gel. This is due to the ability of the gel apparatus to dissipate heat. P=I² R, states that the power produced by the system, P, which is measured in watts, is equal to the current squared times the resistance. The power is manifested as heat and therefore there is a limit on the amount of heat an electrophoresis apparatus can dissipate before the it begins to melt the gel medium.

Because there are so many uses for the polymerase chain reaction (PCR) in molecular biology today such as subcloning DNA fragments, introducing site-directed mutations, sequencing, cDNA cloning, measuring mRNA levels etc., you should understand the basic principles behind it. The PCR method was first described by Mullis and colleagues at Cetus Corp. (Mullis, K. and Faloona, F. 1987. In Methods in Enzymology, Vol. 155 ed. R. Wu, p.355. Academic Press, New York and London) . The discovery of thermostable DNA polymerases greatly facilitated automating the procedure and helped establish PCR as one of the most widely used methods of molecular biology.

Basically PCR is a series of primer extension reactions that also forms the basis for many sequencing and DNA labelling techniques. A primer extension reaction is one in which DNA polymerase synthesizes a new complementary strand of DNA in the 5’ to 3’ direction using a single stranded DNA template but starting or initiating synthesis from a double stranded region. This double stranded region is designated in PCR by the use of two primers or amplimers which are very short pieces of DNA. The two primers are complementary to opposite strands of a region of interest on the DNA. If a double stranded piece of DNA is denatured to two single strands by heating and the primers are then allowed to bind to their complementary regions by cooling the reaction the primers can direct DNA polymerase to extend or synthesize a new complementary strand. This new strand of DNA will contain a complementary region to the other primer in the reaction and can thus be used as a template if another round of denaturation, hybridization and extension are performed. In this way a region of DNA that is flanked by the two primers can be amplified exponentially. These rounds of denaturing, annealing the primer and extension of the primer by DNA synthesis are called cycles in PCR. At what cycle can PCR begin synthesizing a region of DNA exponentially? Can you diagram the first several cycles in a standard PCR reaction? Examine the following:

Third cycle This major PCR product accumulates exponentially

The pH scale is based on K_w , the ionization product of water and is a way of expressing the concentration of H⁺ in the range of acidity between 1.0 M H⁺ and

1.0 M OH^- .

pH = -log [H⁺]

What is the concentration of H⁺ in a solution of 0.1M NaOH?

The Henderson-Hasselbalch equation can be used to make a solution of a desired pH without the use of a pH meter. In biology, this is often done for phosphate buffers. The pK’ of H₂PO₄^- is 6.86

pH = pK’ + log [proton acceptor]

[proton donor]

Due to the plethora of microorganisms found throughout most environments it is oftentimes necessary to employ techniques which insure their exclusion from one’s experimental set-up. This is particularly important when working with growth media as these media are not only usually very good for growing experimental cultures but also undesired cells and microorganisms. Therefore, any minor contamination may quickly become a massive contamination problem. Laminar flow tissue culture hoods are an efficient way to help minimize microorganism contamination. These hoods are designed to allow only sterile air (air which has been funneled through filters) to enter the work area under the hood. However, because tissue culture hoods are large and expensive they are not always readily available. Therefore to sterilize a work area without a hood one can wipe the area with 70% Ethanol then work using a flame, i.e. alcohol lamp, and standard sterile technique.

Fluorescence- The absorption of light (photons) is followed by the emission of light at a longer wavelength (lower energy). Not all excited molecules fluoresce due to vibrational energy. Therefore the probability of fluorescence is denoted by Q, the quantum yield, and is the ratio of emitted to absorbed photons.

Luminescence- The emission of light as a result of a chemical reaction.