unisel UNIVERSITI SELANGOR
FACULTY OF SCIENCE AND BIOTECHNOLOGY (FasBIO) MOLECULAR TECHNIQUES BBS 1231 (LECTURER: MDM. Yasotha a/p Sundaraj)
GROUP 3
Devaraj a/l Ravindran 4111018161 Jeevitha a/p Tana sakaran 4111016861 Sujatha a/p kanniappan 4111009531 Premkumar a/l Subramaniam 4111017391 Theevindran a/l Kesavan 4111017311 Paveanthen a/l Ramachandran 4111023071
INTRODUCTION:
Molecular techniques are important skills to be mastered in order to perform well in areas of biology, chemistry and genetics. Some of the molecular techniques are gel electrophoresis, polymerase chain reaction, DNA scanning and imaging. Since these techniques are advanced in terms of technology used for them, thus the equipments used to carry out these techniques are also advance and complex too. The sophisticated equipments must be handled in a proper manner in order to make a full use of it. Other than that, equipments such as ph meter, multi channel pipette and electron microscope must be calibrated according to their specified timing to ensure their precision, consistency and accuracy.
AIM:
To identify the common equipments used by molecular biologist and understand their functions and their ways of handling.
EQUIPMENTS:
Centrifuge, freezer, gel electrophoresis chamber, light microscope, microwave, pH meter, thermocycler, uv transilluminator, water bath, incubator, shaker, weighing balance, electron microscope, multi channel pipette, HPLC unit- high performance liquid chromatography.
METHOD:
Take a tour around the molecular biology laboratory in the university to identify common equipments in the lab and listen to explanation given by the lecturer on those equipments. Besides that, more equipment should be identified and studied by referring to other sources such as text books and internet.
COMMON INSTRUMENTS IDENTIFIED AND STUDIED: CENTRIFUGE
Centrifuge is an apparatus used to separate components in solution based on their density. This machine has a metal block with holes to hold centrifuge tube and spins at desired speed (rpm) and also for desired period of time. Upon rotation of centrifuge tubes around a fixed axis, the components in the solution sediments according to their densities. Denser substance sediments at the bottom of the tube meanwhile lighter substance tend to reside on top of the denser substance. The capacity of the centrifuge tube ranges from 1.5ml to 15ml or even larger than that depending on the specification of the machine. Bench top centrifuge machine tend to have smaller capacity of centrifuge tube. Centrifuge machine also comes in smaller unit called mini centrifuge. Mini centrifuge is portable, used for short period of time and quick spin.
Centrifuge (brand: ependorf) centrifuge tubes
FREEZER
Freezers are used to store samples at a very low temperature and for a long term. Normal freezers tend to store samples at about -20 degrees Celsius thus those samples could be preserved for a couple of months. Meanwhile, deep freezers are able to store samples at about -80 degrees Celsius and those samples can be preserved for about one year.
Freezer deep freezer
GEL ELECTROPHORESIS
(electrophoresis chamber)
Gel electrophoresis is a method of using gel as a medium to sort out molecules such as deoxyribonucleic acid(DNA) based on size and charge. This is made possible by the movement of charged particle in an electrical field. The gel placed in an electrophoresis chamber is connected to power source, when electric current is applied, the larger molecules move slowly through the gel than smaller molecules. Then ,distinct bands are formed. In order to make those bands to be visible, staining agent is added to the gel. A photograph is also taken under ultraviolet light which makes the bands appear brighter.
(bands of DNA)
LIGHT MICROSCOPE
Light microscope is used to observe tiny objects that are not visible for naked eye. This device has one ocular lens and three objective lens with various magnification powers (x10, x40, x100). It also has coarse and fine adjustment knobs to focus on specimen placed on the stage. The lights source to observe the specimen comes from a light source under the diaphragm. In some less advanced microscope, the light source comes from the reflection of light on the mirror placed under the diaphragm.
MIRCOWAVE OVEN
Microwave oven heats up substance placed in it by passing through non ionizing microwave radiation. The heating compartment of the oven is made up of aluminium and consists of transmitters to transmit microwaves. The heating temperature and timing can be set manually before using it. In laboratory, microwave ovens are used to prepare agarose gel for DNA transfer.
(1000 watt microwave oven)
PH METER pH meter is an electronic instrument used for measuring the pH of a liquid. A laboratory pH meter consists of a special measuring probe connected to an electronic meter that measures and displays the pH reading. pH value is measured based on hydrogen cations in the solution. When the probe is dipped into a solution, the hydrogen ions in it surround the thin-walled glass bulb (probe). The activity of the hydrogen ions causes the probe to produce a small voltage and it will be measured and displayed as pH units by the meter. The meter is similar to voltmeter but the measurements are displayed in pH units instead of volts.
The pH meter has to be calibrated before using because the glass electrode does not give a reproducible electromotive force over longer periods of time. Calibration is performed with at least two standard buffer solutions. The glass probe of the pH meter is the most crucial and sensitive part. It must be kept wet at all times to avoid dehydration of pH sensing membrane and dysfunction of electrode. . After using the pH meter for every measurement, the probe is rinsed with distilled water to remove any traces of the tested solution.
Labrotary pH meter portable pH meter
THERMOCYCLER PCR MACHINE
Thermocycler PCR machine or DNA amplifier is a machine or laboratory apparatus that uses Polymerase chain reaction process to amplify segments of DNA. This machine is very useful when a limited amount of DNA sample need to go through various tests. A thermocycler PCR machine can produce thousands or millions of copies of an original sample to be tested. This machine comes in various designs from various biotech companies, however the function and working principles are the same. This machine has a termal block with holes to insert tubes containing PCR reaction mixtures. The machine then changes the temperature of the block in pre-programmed steps.
thermocyclers of three different brands
UV TRANSILLUMINATOR and CELL DOCUMENTATON SYSTEM
UV transilluminator is a device used to observe bands especially DNA bands found in the agarose gel after being electrophoresised. Some components in the agarose gel fluoresce under UV light, thus the gel will be placed on an UV transilluminator for visualization purpose. This equipment comes in two forms: with and without darkbox design. Darkbox design illuminator has a digital camera to capture the image and save it in a PC unit connected to it. The image captured can be even printed out for other purposes.
UV transilluminator with darkbox
UV transilluminator without darkbox LABORATORY WATER BATH
Laboratory water bath is used to maintain a very stable temperature around any vessels containing reacting substances or chemicals, therefore it can be also used for incubation process. This is made possible by heating and circulating the water in the tank. Other than that, this equipment can also be used to heat substances that cannot be heated by Bunsen burner. The temperature of the water can be set before using it. Water baths can hold temperatures within 100 degrees Celsius.
INCUBATOR
An incubator is a device used to grow and maintain microbiological cultures or cell cultures. The incubator maintains optimal temperature and humidity of the atmosphere inside. Incubators are essential for a lot of experimental work in molecular biology and are used to culture both bacterial as well as eukaryotic cells. Most incubators include a timer which can be programmed to cycle through different temperatures and humidity levels. Incubators can vary in size from tabletop to units the size of small rooms. The simplest incubators are insulated boxes with an adjustable heater, typically going up to 60 to 65 °C. Commonly in microbiological cultures the temperature that is used frequently is 37 ⁰C for the organisms that grows under these conditions (bacteria). Other biological organisms such as yeast the beat temperature used to culture it are 30 ⁰C.
There are many different types and sizes of incubators. Uni-therm incubators, mini incubators, refrigerated incubators, humidity and constant temperature incubators, uni- therm carbon dioxide incubator, unihood incubation hood and cooled incubators are the ones which are mostly used in many laboratory experiments
Uni-therm Incubator Uni- therm Carbon Dioxide Incubator
Uni-hood Incubation Hood Uni-therm Refrigerated Incubator
SHAKER Humidity Incubator Mini Incubator
SHAKER
Shakers are used to culture microorganisms and algae in molecular biology. They shake and vibrate the microorganisms in the beaker that are placed on the shaker. The main purpose of doing this is to stir the solution of the microorganism. The units that are used in shakers are rpm which stands for round per minute. Shakers are mainly powered by an electric motor. Shakers are classified into two which are heated shaker and unheated shaker. Both this shaker owns different characteristics but with same function. It has indexing device and timer. The frequency of this unheated shaker is from 40 to 200 rpm with the amplitude of 20mm. Another unheated shaker has horizontal shaking motion. It also has indexing device and timer as the other one. The frequency of this type is 50 to 1100 rpm with amplitude of 3mm.
Heated shaker are the shakes with heated platform. It’s a microprocessor controlled thermo shaker for 4 micro liter plates with -65°C RT and 3mm orbit. Heated shaker, shakes frequently throughout the particular period of time.
Shaker WEIGHING BALANCE
Weighing balance is used to measure mass to a very high degree of precision and accuracy. The measuring pan of a high precision weighing balance are placed inside a transparent enclosure with doors to avoid dust from being collect and from any air currents in the room do not affect the balance's operation. The use of a vented balance safety enclosure, which has uniquely designed acrylic airfoils, allows a smooth turbulence-free airflow that prevents balance fluctuation and the measure of mass down to 1 μg without fluctuations or loss of product. Also, the sample must be at room temperature to prevent natural convection from forming air currents inside the enclosure, affecting the measure of mass.
Weighing precision is achieved by maintaining a constant load on the balance beam, by subtracting mass on the same side of the beam to which the sample is added. The final balance is achieved by using a small spring force rather than subtracting fixed masses.
Weighing Balance
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY UNIT – HPLC UNIT
HPLC unit is a tool used to carry out a highly improved liquid chromatography. In a normal liquid chromatography tool, a solvent is allowed to drip through a column under the influence of gravity but in a HPLC, solvent is forced through under high pressures up to 400 atmospheres which makes it much faster. The components of this HPLC unit are solvent reservoir, a pump to produce high pressure, HPLC tube, detector and a processing unit to display it. This equipment allows a better separation of the components of the mixture. The injection of sample into the HPLC tube is an automated process.
HPLC – unit
CONCLUSION:
Common equipments used by molecular biologist, their function and the ways to handle them are identified and learned.
REFERENCE:
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