Basic Instrumentation

Abstract
As a student of cell molecular biology, it is important to be knowledgeable and
familiar with proper care and use of the laboratory tools and equipment.
Researching and compiling for the proper description and uses of the tool and
equipment, which would be used in cell molecular biology.
(University of the Philippines Manila- Biology Department)

Keywords:
Bio ref., UV/Vis spectrophotometer, Photo documentation system, Micropipettors, Thermal
cyclers, Gel electrophoresis system(horizontal, vertical, power supply, fast blast staining
system), shaker/agitator, Vortex mixer, Microcentrifuge, water bath, Distilling apparatus,
Autoclave/sterilizer, Ice shaver, Parafilm

Introduction
Cell and Molecular Biology is an interdisciplinary field that bridges chemistry,
structure and biology understanding life and processes in a molecular level. The
basic mechanisms that allow cells to have differentiated properties and coordinate
the activities forming systems essentials in defining a living cell.
(www.scripps.edu/research/cmb/)
Students of cell molecular biology, should know the importance of being
knowledgeable and familiar with proper care and use of the laboratory tools and
equipment.
These knowledge would help lengthen the life of the laboratory tools and
equipment, and helps student to get positive results with their experiments. This
introduction would serve as one of their foundation in cell molecular biology.
(University Of the Philippines Manila- Biology Department)

Materials and Methods

Bio Ref.

The use of bio ref. is to store volatile, noxious and air sensitive materials, its not
uncommon for the atmosphere inside the unit to be saturated with chemical vapors.
It is used to preserve and cool specimens from the laboratory

There are four types of laboratory refrigerators.

Explosion proof refrigerators designed for storing flammable liquids

and hazardous chemical substances. It lacks electrical equipment that
prevents fire caused by sparks in the storage area, and is safe to use with
combustible materials.
Lab fridges using a digital display it is designed to maintain consistent
temperatures and monitor the temperatures. It needs and include lockable
easy-to-clean sections for they are general laboratory refrigerators. They
also used in cooling samples and for preservation.
Blood bank refrigerators it complies with all American and European
regulations. Reliability along with the ability to monitor temperatures is
critical for this type, also they have separate compartments for storing
different sample types.
Chromatography refrigerators designed for research experiments.
Best used in laboratories for medical samples and procedures requiring
precise temperature settings and stability.

UV/Vis Spectrophotometer

UV/Vis/NIR spectroscopy are used in studying the optical properties of photovoltaic

cells. The equipment is required to measure the various phenomena involving
(reflectance, transmittance, and absorbance). to determine the selective reflectivity,

it involves calculation and studies based on a silicon cell.( Catherine Tams

PerkinElmer, France Nicolas Enjalbert INES)
Electronic transitions are causes of Ultraviolet (UV) and Visible (VIS) light. Higher
energy orbital, the energy absorbed excites an electron into an empty, when a
molecule absorbs UV-VIS radiation. The absorbance of energy can be plotted
against the wavelength to yield a UV-VIS spectrum. UV-VIS spectroscopy can also
be used in detection of eluting components in high performance liquid
chromatography (HPLC), determination of the oxidation state of a metal center of a
cofactor (such as a heme), or determination of the maximum absorbance of a
compound prior to a photochemical reaction. The shape of the peak(s) and the
wavelength of maximum absorbance (lmax) both gives information about the
structure of the compound.
Using a UV/Vis spectrophotometer
1. Double-click the HP UV/Vis Online icon to open chemstation.
2. when prompted to enter a password, Press the Cancel button
3. To turn on the lamp Use the Instrument dropdown menu. There is only one
lamp on the 8432. It serves as both the visible and UV light source. There are
two lamps in the 8453. Turn on the deuterium lamp for scanning the UV
region and the tungsten lamp for visible scans.
4. At the top right of the Chem Stations window, make sure the Mode is set to
standard.
5. In the Task window, select Spectrum/Peaks from the dropdown menu if it is
not already selected. Choose the Setup icon in the Task window. The data type
should be set to Absorbance. For a visible spectrum, make the lower wavelength
limit 400 nm and the upper 800 nm. For scanning the ultraviolet region, make the
limits 200 and 400 nm.
6. In the Sampling window, select Manual from the dropdown menu if it is not
already selected.
7. Fill a cuvette with solvent and place the cuvette into the sample holder. Lock
the cuvette into place using the lever on the side of the holder.
8. Obtain a background spectrum by clicking the word Blank in the Sampling
window. If a Last Blank Spectrum window appears, simply close it.
9. Remove the cuvette from the sample holder and fill the cuvette with your
sample. Return the cuvette to the sample holder.
10. Obtain a spectrum by clicking Sample in the Sampling window.
11. The spectrum should appear in the display. If it is satisfactory (absorbance <1
for most intense peak), then select the Overlaid Sample Spectra window and print
your spectrum.
Error Messages: If you see a red error message bar at the bottom of the Chem
Stations window, read the text and correct the problem.
To export the data to Excel:
12. Select the spectrum itself by clicking on the curve. (Data point boxes should
appear on the curve once it is selected.) Use the File dropdown menu and choose
Export Selected Spectrum as then CSV format. Save your spectrum to a floppy
disk. What's the advantage? You can easily determine lmax, even for a shoulder, by

viewing the data points in Excel. You can also format the graph in Excel and easily
insert it into a Word document.
When you are done:
13. If others are waiting for the instrument, click the Clear icon on the toolbar to
clear your spectrum from the window. If no one is waiting to use the instrument
immediately, turn off the lamp(s) and close the HP software.
14. Dispose of any waste and clean your cuvette.
Micropipettes

The micropipettors are used to transfer small amounts of liquids. The scales on
micropipettors are in microliters (1000l = 1 ml). Micropipettors are also used in
conjunction with disposable (often sterile) plastic tips. The following is an illustration
of a micropipettor:
Using a Micropipette
1. Never exceed the upper or lower limits of these pipettors. The limits are: P20:
0.5 to 20.0 l P200: 20 to 200 l P1000: 200 to 1000 l
2. Set the desired volume by turning the centrally located rings clockwise to
increase volume or counterclockwise to decrease volume. P20: Maximum
volume 20 l. Accurate between .5 l and 20 l. Numbers on the
micropipetter (typically black-black-red) are read as XX.X l. The change in
color indicates the position of the decimal point. P200: Maximum volume 200
l. Accurate between 20 l and 200 l. Numbers on the micropipetter (one
color) are read as XXX l. P1000: Maximum volume 1000 l (= 1 ml).
Accurate between 200 l and 1000 l. Numbers on the micropipetter
(typically red-black-black) are read X.XX ml. Note that this micropipetter
reads milliliters while the other two read microliters.
3. Place a tip on the discharge end of the pipettor. NOTE: If sterile conditions are
necessary do not allow the pipet tip to touch any object (including your
hands).
4. The plunger will stop at two different positions when it is depressed. The first
of these stopping points is the point of initial resistance and is the level of
depression that will result in the desired volume of solution being transferred.
Because this first stopping point is dependent on the volume that is being
transferred, the distance you have to push the plunger to reach the point of

5.

6.

7.

8.

initial resistance will change depending on the volume being pipetted. The
second stopping point can be found when the plunger is depressed beyond
the initial resistance until it is in contact with the body of the pipettor. At this
point the plunger cannot be further depressed. This second stopping point is
used for the complete discharging of solutions from the plastic tip. You should
not reach this second stop when drawing liquid into the pipettor, only when
expelling the last drop. Before continuing, practice depressing the plunger to
each of these stopping points until you can easily distinguish between these
points.
Depress the plunger until you feel the initial resistance and insert tip into the
solution, just barely below the surface of the liquid and not as deep as
possible.
Carefully and slowly release plunger. NOTE: If the solution you are pipetting is
viscous, allow the pipet tip to fill to final volume before removing it from
solution to avoid the presence of bubbles in the plastic tip which will result in
an inaccurate volume.
Discharge the solution into the appropriate container by depressing plunger.
This time, depress the plunger to the point of initial resistance, wait one
second, and then continue pressing the plunger as far as it will go in order to
discharge the entire volume of solution.
Remove tip by pressing down on the tip discarder.
(https://www.wou.edu/las/physci/ch462/How%20to%20Use%20a
%20Micropipettor.pdf)

Thermal Cycler (PCR)

Using polymerase chain reaction Thermocyclers, or thermal cyclers, amplifies

DNA and RNA samples. The thermocycler raises and lowers the temperature of
the samples in a holding block in discrete, pre-programmed steps, allowing for
denaturation and reannealing of samples with various reagents. Cloning,
sequencing, expression analysis, and genotyping are downstreamed applications
Amplified by genetic material.
Shaker/Agitator

Laboratory shakers and rotators are used to blend or agitate samples within
flasks or tubes, it contains and consist of housing motor and control panels, upon
which an agitation platform is attached. It has a platform that has simple
grooves to support flasks and tubes horizontally as the device moves, or it may
have basket style holders that keep the sample holders upright.

Types of shaker/agitator

Vortex Mixer

Reciprocating which moves alternately backward and

forward.
Rocking this device do a rocking or seesaw motion.
Rolling this device moves from side to side with a slight
upward, then downward tilt.
Rotating shakers turn about an axis and function similarly to
centrifuges, but they do not reach the same speeds.
Orbital these are the most common variety of laboratory
shakers. Creates an orbital (horizontal circular) shaking
motion sufficient for mixing liquids in flasks and conducive to
culturing cells. Most incubator style shakers prefer this
design.
Wrist or hand motion laboratory shakers mimics the swirling
motion of hand mixing. Instead of cradling flasks or tubes
within the agitation platform, it has long arms attached to the
housing, which swing and swirl when the device is turned on.

The main purpose of a Vortex Mixer is to mix small vials of liquid. Its a simple
device that has an electric motor with a drive shaft which is vertically oriented and
also attached to a cupped rubber piece mounted slightly off center. It works by
motor with a rubber piece doing a circular motion so as the test tube is placed on it
the rubber will rotate it and so as its content. Like other laboratory equipment it has
optional speed and settings. It is used in cell culture and microbiology laboratories
to help cells settle, or it can also be used to mix reagents or experiments.
Microcentrifuge

A microcentrifuge, also called a microfuge, is a very important laboratory

equipment; it is used to spin small (2 ml or less) liquid samples at high speeds
(generally tens of thousands times g-force). In many biological applications small
samples of Centrifugation is important, for example pelleting nucleic acids or
proteins from solution, microfiltration of small aqueous samples or even in simple
gathering the last precious drops of liquid into the bottom of the tube.
microcentrifuges are usually small, compact benchtop centrifuges with a small
footprint, available in refrigerated or non-refrigerated (ventilated) models. (Caitlin
Smith, 2011)

Water bath

A water bath is a device used to maintain constant water temperature. It is also

used as incubation in microbiological laboratory.
First we must check whether the water bath is on turned on, afterward we must set
it at the right temperature, and filled with water. We should use distilled water in
filing water, and during an experiment we should constantly check the temperature
of the water bath to make sure that it is maintaining the right temperature.
Water Bath Controls:
Temperature Control: All water baths have a control to set temperature. This
control can be digital or a dial. Often there is an indicator light associated with this
control. When the light is on the water bath is heating. When the water bath
reaches the set temperature, it will cycle on and off to maintain constant
temperature.
Safety Control: Most water baths have a second control called the safety. This
control is set at the maximum temperature the water bath should attain. It is

usually set just above the temperature control. Often an indicator light is associated
with the safety control. If the water bath reaches the temperature that the safety
control is set at, the light will go on. It will be impossible for the water bath to heat
higher than the safety setting even when the temperature setting is higher. If your
water bath stays a temperature lower than the temperature control setting, try
increasing the safety control setting.
Shaking Control: Shaking water baths have additional controls for shaking. The
shaking mechanism can be turned on or off. The speed of shaking can also be set.
Cultures grown in liquid media are often shaken to allow constant mixing with air
and oxygen with the culture. (Mouse over picture to see a large image of a shaking
water bath) (Microeguide, 2010)
Distilling Apparatus

Distilling apparatus are device used to separate liquid mixtures from fractions of
different compositions by means of simple distillation. It brings the mixture to boil in
a distillation vat, or still, then the vapors are drawn off to a cooler-condenser.
Distilling apparatus are used in the food industry for producing essential oils,
aromatic alcohols, and cognac alcohols; they are also used in petroleum refining
and wood chemistry.
(Anoshin, I. M. Teoreticheskie osnovy massoobmennykh protsessov pishchevykh proi
zvodstv. Moscow, 1970.Kasatkin, A. G. Osnovnye protsessy i apparaty khimicheskoi
tekhnologii, 9th ed. Moscow, 1973.)
Autoclave/Sterilizer

An autoclave is a large pressure cooker, use to sterilize; it uses steam under

pressure as the sterilizing agent. It uses High pressures that enables the steam to
reach high temperatures, thus increasing its heat content and killing power. The
latent heat of vaporization is where Most of the heating power of steam came from,
This is the amount of heat required to convert boiling water to steam. This certain
amount of heat is large compared to that required to make water hot. Achieving
high and even moisture content in the steam-air environment is important for
effective autoclaving. The ability of air to carry heat is directly related to the amount
of moisture present in the air. The moister there is, the more heat it can carry, so
steam is one of the most effective carriers of heat. Therefore Steams are an efficient
killer of cells, and the coagulation of proteins.
How it works; the steam would enters the chamber jacket, then passes through an
operating valve and enters the rear of the chamber behind a baffle plate. It would
flow forward and down through the chamber and the load, exiting at the front
bottom. A pressure regulator maintains jacket and chamber pressure at a minimum
of 15 psi, the pressure required for steam to reach 121C (250F). Overpressure
protection is provided by a safety valve. The conditions inside are thermostatically
controlled so that heat (more steam) is applied until 121C is achieved, at which time
the timer starts, and the temperature is maintained for the selected time. (Howard
Judelson 6/28/04)
Ice Shaver

Ice shaver could be a Hand operated or motor driven floor or bench mounted
machine that has a rotating plate or wheel and a sharp knife that produces ice like
snow when forced against the face of a cake of ice. Also called Snowcone Machine.
(www.fcsi.org)
Parafilm

Parafilm M is a moisture-resistant thermoplastic used in research ,mostly in clinic

and industrial laboratories work. This laboratory film can be used to prevent
contaminations and evaporation or to avoid spills. Parafilm M is self-sealing,
flexible, odorless, semi-transparent and it can extend to irregular .form or shape
cover providing a reliable barrier. It has different sizes, Parafilm M can be useful in
electron microscopy droplet staining and thin-film grid coating. It can also serve as a

non-slip surface on trays and shelves for instruments or containers.(

www.tedpella.com)
Photo Documentation system

Photo documentation system is used to document photos of experiments either

printout or software copy that can be treated and edited in a computer, it also helps
us in monitoring our experiment more accurately and easier by providing a not only
written data but also with images. (DOC.PRINT is supplied with a software
PHOTOCAPT which does not require an additional card to incorporate
in the computer). Here are some of the activities that we can apply this photo
documentation system:
Nucleic acids detection and Proteins detection etc.
(www.labolan.es)
Gel Electrophoresis system
A gel electrophoresis apparatus allows the researcher to maintain a uniform electric
field across the gel, provide cooling to prevent thermal artifacts, and allows access
to the gel for sample loading and monitoring the run. There are Two types of
apparatus commonly used, the vertical and horizontal. Vertical gel systems are
further subdivided into slab gels and tube gels. In general, agarose gels are run in
the horizontal format, while acrylamide gels are run vertically.

Horizontal Gel Electrophoresis

In a simplest form, a horizontal gel apparatus consists of a box which is divided into
two compartments using a platform right in the middle. The gel is placed on this
platform, and well add buffer until the gel is completely submerged. There are
Electrodes in every compartment that supplies the electric field. We should control
the thickness of these to have a positive result, this is the result from current flows
of both the gel and the buffer over the gel. The Cooling effect is provided by the
buffer which surrounds the gel, and this buffer is often recirculated to prevent the
development of a pH gradient and also to aid in temperature control. To Access the
gel we should go to the overlaying buffer. We load the Samples through this buffer
layer, it has a clear lid so that we could monitor the run. One major limitation of the
horizontal apparatus is that, since the two compartments are connected by a layer
of buffer, it is not possible to use discontinuous buffer systems. Another limitation is
that the gels are cast in trays which are not covered. Because atmospheric oxygen
has full access to the upper surface of the gel, acrylamide will not polymerize in this
system. Horizontal systems are primarily used to run agarose gels which are run in
continuous buffer systems and are not affected by O 2. For agarose gels, the
simplicity and ease of use of the horizontal system make this system the best
choice. (www.nationaldiagnostics.com)
Vertical Gel Electrophoresis

Vertical apparatus are usually used for sequencing. This system shows the
components common to all vertical slab systems. The gel is placed between two
glass plates that is separated by spacers, usually less than 2mm thick. The gel is
mounted in the system so that the top is in contact with the negative electrode
chamber, and the bottom is in contact with the positive electrode chamber.
Compare to the horizontal system, its connection to the buffer chambers is through
the gel. This allows precise and reproducible control of the voltage gradient.
Because of the high resistance of the thin gel, the apparatus must have provisions
for cooling. The front of the gel cassette is exposed to the air, while the back of the
gel is held against a metal plate which dissipates heat rapidly. In some systems, the
upper buffer chamber extends almost to the bottom of the gel, and the upper buffer
is used for cooling. The relatively small amount of current carried through the gel
means that buffer recirculation is generally not required. In general, vertical slab
gels are loaded through the top, under a layer of buffer. The gels are monitored
during the run through the front glass plate. The fact that the body of the gel in
these systems cannot be accessed until the end of the run can be an
inconvenience. Some sample recovery techniques used on horizontal gels are not
available for vertical gels. However, the resolution and reproducibility of vertical
polyacrylamide gels more than compensate for this. (www.nationaldiagnostics.com)
Power Supply

This high voltage power supply serves as a connection to an electrophoresis tank

setting up an electric field between the two electrodes. Using (+) anode the DNA
samples that we loaded into an agarose gel move through the gel, with the agarose
gel matrix separating the DNA molecules by size. Like other
apparatus Electrophoresis power supplies also has optional adjustment settings for
output voltage depending on the gel tank size, to get the optimum result.
(http://www.instructables.com)
Fast Blast Staining facility

Fast Blast DNA stain serves as an in-gel stain, In an agarose gel we incorporate the
fast blast DNA stain along with the electrophoresis buffer so that it would stain the
DNA as it runs through the gel. This in-gel staining method is quick but less accurate
method of staining bands. This method is not ideal for experiments in which
desirable bands are determined by precise molecular weight since the DNA bands
would appear fuzzy. (www.bio-rad.com)

Questions:
1.) How is a spectrophotometer used to determine DNA concentration and
purity?

Both can be determined using ultraviolet light by measuring it. Depending on

the length of wave DNA would absorb UV. Using a Nano Drop
spectrophotometer, well measure the wavelengths and determine the
absorption spectrum. Well observe when the maximum absorbance would will
occur and determine how much UV the DNA would absorb.
2.) The speed of micro centrifuge can be measured in rpm or x g. how do you
convert between this two units?
The simple function of rotation speed of the centrifuge and radius of its
rotation, determines the force exerted on the particles in a centrifuge, that
gives this equation to convert:
RCF or G-force= 1.12 x R x (RPM/1000); in millimeters we measure
the R or the radius of rotation.
3.) In PCR, what is the relationship between the DNA templates melting point
and the annealing temperature? Explain your answer briefly.
In PCR, DNA amplification undergoes repeating cycles of three temperature:
1st the (dsDNA) or double stranded DNA template is denatured. 2 nd the
oligonucleotide primers are annealed to the (ssDNA) or singe stranded DNA
template, a primer is designed to anneal in a specific area on the left side of
one of the DNA strand and same to the right as a complementary strand of
DNA. 3rd the DNA polymerase would extend primers to duplicate the DNA
fragments between primers. With each cycle the DNA fragments are
amplified.

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Basic Instrumentation
Exercise 1

Ambawas,Jaylou
Bacalando,Jose
Estopel,Honie Grace
Hulog,Cha
Mendez,Hyasmin
Tubato,Aza Freya Lan