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Different Laboratory Equipment used in Toxicology and Molecular Biology
This document describes various types of laboratory equipment used in toxicology and molecular biology. It discusses personal protective equipment, volume measuring tools like beakers and volumetric flasks, microscopes for identification, analyzers like microplate readers and electrophoresis apparatus, chromatography equipment, and other tools like refrigerators, sterilizers, and centrifuges. The functions, components, principles, and applications of these different pieces of equipment are explained.
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Different Laboratory Equipment used in Toxicology and Molecular Biology
- 1. DIFFERENT LABORATORY EQUIPMENTUSED IN TOXICOLOGY AND MOLECULAR BIOLOGY Muhammad Kamran M.Sc(Hons) Agri.Entomology University College of Agriculture, University of Sargodha Pakistan
- 2. Contents 1. Personal ProtectiveEquipment (PPE) 2. Volume Measuring Equipment 1. Non-volumetric containers 2. Volumetric labware 3. Equipment used for identification 1. Hand lens 2. Light Microscope 3. Inverted Microscope 4. Fluorescence Microscope 4. Analyzing Equipment (Analyzers) 1. Microplate Reader 2. PCR apparatus 3. Electrophoresis apparatus 4. Spectromer (UV/VIS ) 5. Chromatography Equipment (GC,TLC, HPLC) 5. Other Equipment 1. Laboratory Refrigerators and Freezers 2. Instrument Sterilizers 3. Centrifuge
- 4. Chemical-resistant coveralls Chemical-resistant gloves Chemical-resistantfootwear Chemical-resistant hood or wide-brimmed hat
- 5. Chemical-resistant apron Chemicalfume hoods are used to protect workers from exposure to volatile chemicals.
- 7. o For mixingand storing solutions and are generally not calibrated. o Instead, the measurements, or graduations, on the side represent approximations of liquid capacity. 1.Non-volumetric containers Glass beaker Erlenmeyer flasks
- 8. 2. Volumetric labware oMeasure exact volumes of liquid substances. o Volumetric labware is denoted with the capacity it is calibrated to hold as well as the letters TC or TD. o Volumetric flasks prepare solutions of a specific concentration. o Graduated cylinders are used to measure volumes of liquid above 50 mL. Volumetric flasks Graduated cylinders
- 9. o Serological pipettes:Generally used to measure and deliver volumes in the range of 0.1 to 50 mL. o Syringes: The use of syringes is the most frequent method for transferring liquid chemicals or solvents in the laboratory.
- 10. o Micropipettors: Forvolumes of 0.2 microliters to 5 mL, micropipettors should be used. o Glass Hamilton syringes: When plastic pipette tips are not compatible with the liquid to be measured, glass Hamilton syringes are an alternative for accurate measurement of volumes in the microliter range.
- 12. o A magnifyingglass is a convex lens that is used to produce a magnified image of an object. o The lens is usually mounted in a frame with a handle o Hand lenses is used for identifying gems, crystals, rocks, insects, leaves or spiders from 5-30X magnification. Hand lens
- 13. The light microscope Thelight microscope is an instrument used for magnifying research specimens. Allow to view objects at 1000 times their original size.
- 14. Inverted microscope An invertedmicroscope is a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up.
- 15. The main componentsof the light microscope
- 16. Applications • Light microscopyhas the potential to visualize a wide range of specimens. • various configurations of the compound microscope exists to suit many different applications.
- 17. Fluorescence microscopes A fluorescencemicroscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances
- 18. Principle Components • Maincomponents of the fluorescent microscope overlap greatly with the traditional light microscope. • The 2 main differences: o The type of light source. o The use of the specialized filter elements.
- 19. Applications • Imaging ofproteins • To integrate the code for a fluorescent protein such as green fluorescent protein, or GFP, into the DNA of an organism. • To investigate tumor cells • Fluorescence Speckle Microscopy to study movement and turnover kinetics of the protein.
- 21. Microplate reader o Themicroplate reader allows for a variety of experiments to be performed and measured simultaneously. o Microplate readers can make absorbance, fluorescence and luminescence measurements. Multiplate readers are used o To quantify protein o Gene expression o Various metabolic processes such as reactive oxygen species and calcium flux.
- 22. Components of aMicroplate Reader • Microplate readers come in different shapes, sizes and set-ups. • Multiwell plates: used to hold the samples that are measured by the machine. • The loading tray: used to bring the 96-well plate into the machine. • A computer interface: to operate the plate reader and control its settings and parameters.
- 23. Applications • These areused in drug discovery, research, bioassay validation, and biopharmaceutical manufacturing. • ELISAs, • Protein and nucleic acid quantification or enzyme activity assays • Bradford assay • Fluorescent-based assays • Luminescent assays • High-throughput assays
- 24. PCR machine isused for an in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence PCR Machine
- 26. Major Components • TemplateDNA • Oligonucleotide primers • Thermostable DNA polymerase • MgCl2 • dNTP’s • Buffer • Working concentrations • KCL • Tris-HCl • NaCl2 (sometimes) • Thermocycler • Detectors Primers DNA template Buffer + + A C T G MgCl2
- 27. Principle Typically, a PCRis a three-step reaction. o Denaturation of DNA o In the first step of PCR, the sample is heated to 95–98°C, which denatures the double-stranded DNA, splitting it into two single strands. o Primer hybridization ( Annealing) o In the second step, the temperature is decreased to approximately 55–65°C, allowing the primers to bind, or anneal, to specific sequences of DNA at each end of the target sequence, also known as the template. o DNA synthesis ( Primer extension) o In the third step, the temperature is typically increased to 72°C, allowing the DNA polymerase to extend the primers by the addition of dNTPs to create a new strand of DNA, thus doubling the quantity of DNA in the reaction.
- 28. Step 1: Denaturation dsDNA tossDNA Step 2: Annealing Primers onto template Step 3: Extension dNTPs extend 2nd strand Vierstraete 1999 extension products in one cycle serve as template in the next 28
- 29. Applications • Genome mappingand gene function determination • Biodiversity studies ( e.g. evolution studies) • Diagnostics ( prenatal testing of genetic diseases, early detection of cancer, viral infections, mutation detection...) • Detection of drug resistance genes • Forensic (DNA fingerprinting) • Microarrays, molecular cloning, recombinant DNA research.
- 30. Detection of amplificationproducts • Gel electrophoresis • Sequencing of amplified fragment • Southern blot • etc... 30
- 31. Gel Electrophoresis Equipment Electrophoresis apparatusis used for the separation of charged molecules in an applied electric field. 31
- 32. Major components o Boxto hold the gel o Comb to create small wells in the agarose gel to put the DNA sample into at the beginning of the gel o Positive and negative electrodes to create the electrical current o Power supply o Gel photo imaging system 32
- 33.
- 34. Applications of GelElectrophoresis DNA can be separated by electrophoresis to: • Visualize bands of a molecular marker to genotype individual • Verify amplification by PCR or sequencing reactions • Check the quality and quantity of genomic DNA after DNA extraction • Used for investigating the DNA • To investigate various binding modes of small molecules to supercoiled DNA • Complex mixtures can be separated to very high resolution by this process (Sheehan, D.; 2000).
- 35. Chromatography Equipment (TLC) The solventtravels up plate via capillary action and the compounds separate depending on compound solubility. Detection: Color changes or by using UV light to observe bands. (HPLC) A highly improved form of column chromatography. Solvent is forced through under high pressures of up to 400 atmospheres. That makes it much faster. (GC) Gas chromatography is a technique used for separation of volatile substances, or substances that can be made volatile, from one another in a gaseous mixture at high temperatures. Detection: By comparing retention times of peaks in a sample to retention times for standards.
- 42. HPLC Detectors oUV/Vis oRefractive index oFluorescence oEvaporativelight scattering (ELSD) oMS oDiode Array Detector (DAD) 42
- 43. Application of HPLC 1.Pharmaceuticals industry • To control the drug stability • Quantity of drug determination from pharmaceutical dosage forms, ex. Paracetamol determination in panadol tablet • Quantity of drug determination from biological fluids, ex: blood glucose level 2. Analysis of natural contamination 3. Forensic test - Determination of steroid in blood, urine & sweat. - Detection of psychotropic drug in plasma 4. Clinical test 5. Food and essence manufacture 43
- 44. Spectrometers Spectroscopy is based,principally, on the study of the interaction between radiation and matter. This interaction causes in the atom an electronic transition from a lower energetic level, m, to a higher level, l, occurring energy absorption from the atom equal to the energy difference between both levels, El - Em.
- 45. UV and VisibleSpectroscopy o UV-VIS spectroscopy is used to detect the presence of chromophores like dienes, aromatics, polyenes, and conjugated ketones, etc o It uses light in the visible and adjacent ranges. o The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. o This technique is complementary to fluorescence spectroscopy.
- 46. Application o Quantitative Analysis oRate Measurements o Analysis of Mixtures o Chemical Reaction o Biochemistry o Molecular biology. o Used as a detector for HPLC.
- 48. • Measuring tapes •Insect-proof containers • Scissors • Fine forceps • Fine pointed brush
- 49. o Instrument sterilizers oAnalytical balance can measure down to ten thousandths or even hundred thousandths of a gram.
- 50. Laboratory refrigerators andfreezers
- 51. Centrifuge A centrifuge isused to separate components of a complex mixture.
- 52. Principle component o Therotor, which is the moving part that spins at high speeds. o Many centrifuges will have a refrigeration unit that allows the internal temperature to be controlled during the spin.
- 53. Applications The centrifuges canbe used: • To purify biological specimens like animal cells. • Useful for quickly isolating one cell type from another, or for isolating individual organelles. • The ultracentrifuge can spin in excess of 70,000 rpm, which makes it well suited for the isolation of small particles, like DNA or viruses.