Equipment related to molecular biology

This presentation slide is about the equipment related to molecular biology and the uses and the basic principles of them

1. Laboratory Equipment related to Molecular Biology M. N. F. Nashath Temporary Demonstrator Department of Biosystems Technology Faculty of Technology South Eastern University of Sri Lanka 1

2. ILOs: To list out various laboratory equipment related to molecular biology To identify different laboratory equipment related to molecular biology To describe basic principle and uses of those equipment 2

3. Basic laboratory equipment and consumables in molecular biology Equipment Autoclave Incubator Microwave oven Shaker Water bath Centrifuge Vortex mixer Thermocylers /PCR machine Gel electrophoresis apparatus Gel documentary system Particle bombardment/Gene gun Consumables Micropipettes Glass wares Nitrile gloves Tubes Eppendorf tubes Centrifuge tubes 3

4. Pipette / Micropipette Usage: to measure and deliver accurate volumes of liquid Volume range: 0.2-2 µl 1-10 µl 2-20 µl 20-100 µl 20-200 µl 100-1000 µl 4

5. Pipette holder Pipette tips Pipette tips box 5

6. Unit conversions 1 l = 1000 ml 1 ml = 1000 µl 1 kg = 1000 g 1 g = 1000 µg 1 ml = 1 cm 3 1 m 3 = 1000 l 1 ppm (parts per million) = 1 mg/l = 1 µg/ml 6

7. Molar, % and X solutions A molar solution is one in which 1 liter of solution contains the number of grams equal to its molecular weight 1 M = 1 moles/liter Ex. To make up 100 ml of a 5M NaCl solution ( MW of 58.456 g/ mol ) = 58.456 g/ mol × 5 moles/l × 0.1 l =29.23 g in 100 ml 7

8. Percentage solutions percentage (W/V) = weight (g) in 100 ml of solution percentage (V/V) = volume (ml) in 100 ml of solution Ex. To make a 0.7% solution of agarose in TBE buffer, weight 0.7g of agarose and bring up volume to 100 ml 8

9. “X” solutions Many enzyme buffers are prepared as concentrated solutions Ex: 5X or 10X (5 or 10 times the concentration of the working solution) and are diluted such that the final concentration of the buffer in the reaction is 1X Ex: To set up a restriction digestion in 25 µl add 2.5 µl of 10X buffer, other reaction components and water to a final volume of 25 µl 9

10. Dilution Factor Ratio of the volume of the initial (concentrated) solution to the volume of the final (dilute) solution Ex: What is the dilution factor if you add a 0.1 ml aliquot of a specimen to 9.9 ml of diluent ? Final volume = aliquot volume + diluent volume =( 0.1 + 9.9) ml =10.0 ml DF = 10/0.1 = 100 10

11. Volumetric flask Conical flask Beaker Test tube Graduated cylinder 11 Glass wares….

12. Motar and Pestle Eppendorf Tubes Centrifuge Tubes Nitrile gloves 12 Others….. Glass rod

13. Autoclave Usage: to sterilize plastic, glass ware, culture media and wastages using high levels of heat and pressure Destroys harmful organisms and pathogens Conditions: Temperature: 121 o C Duration : 30 minutes Pressure : 1.06 kg/cm 2 (15 psi) 13

14. Incubator Usage: to provide a controlled, contaminant-free environment for cell and tissue culture Maintains optimal Temperature Humidity CO 2 and O 2 content 14

15. Microwave oven Usage: to heat laboratory samples and to prepare solutions Uses microwaves (radio waves) to heat the samples 15

16. Water bath Usage: to incubate samples in water at a constant temperature over a long period of time Can be used up to 99.9 o C Shaking water bath has extra control for shaking, which moves liquid around 16

17. Shaker Usage: to mix small volume of solutions 17

18. Liquid Nitrogen Usage: to easily crush the tissues by freezing to store cells at low temperature to deactivate harmful chemicals and natural enzymes (nucleases) It is Nitrogen in liquid state at low temperature ( -195.79 o C ) 18

19. 19

20. Vortex mixer Usage: mixing small vials of liquids 20

21. Centrifuge Usage: to separate particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed Sedimentation principle Denser particle – bottom Low-density substances – top 21

22. 22

23. Rotates about an axis resulting in strong force perpendicular to the axis 23

24. Types of rotors Fixed angle rotors Swinging bucket rotors / horizontal rotors Vertical rotors 24

25. Relative Centrifugal Force (RCF) – g Force It is the measure of the strength of rotors This is the force exerted on the contents of the rotor as a results of the rotation RCF is the perpendicular force acting on the sample that is always relative to the gravity of the earth 25

26. Types of centrifuge Bench top centrifuge I t is a compact centrifuge commonly used in clinical and research laboratories 26

27. Low-speed centrifuge Used in laboratories for the routine separation of particles Speed: 4000-5000 rpm Usually operated under room temperature 27

28. High-speed centrifuge Commonly used in more sophisticated laboratories with the biochemical application Speed: 15,000-30,000 rpm Systems for controlling speed and temperature are present 28

29. Micro centrifuge U sed for separation of samples with smaller volumes (0.5 – 2 µl) Speed: 12000 – 13000 rpm U sed for separation of cell organelles like nuclei and DNA 29

30. Refrigerated centrifuge These centrifuges are provided with temperature control ranging from -20 o C to -30 o C Have a temperature control unit Used to separate yeast cells, chloroplasts and erythrocytes Speed at 30,000 rpm 30

31. Ultracentrifuge Operates at extremely high speed at 150,000 rpm Used to separate much smaller molecules like ribosomes, protein and viruses Refrigeration systems are present 31

32. DNA Extraction Kits Many commercial kits are available to isolate DNA from a variety of biological materials Components Wash buffer Binding buffer Elution buffer DNA spin columns 32

33. Advantages DNA can be extracted more conveniently Contaminants like nuclease and proteins are completely removed Avoiding precipitation and use of organic solvent Damage to DNA is minimized The isolated DNA is ready for use in various application 33

34. Spectrophotometer Usage: to determine the average concentration of nucleic acids (DNA or RNA) in a sample Concentrations are obtained by measuring the amount of light absorbed by the solution in a cuvette cuvette 34

35. Types of spectrophotometer UV and visible spectrometer Infrared (IR) spectrometer Atomic absorption spectrometer Atomic emission spectrometer X-ray fluorescence spectrometer Mass spectrometer 35

36. 36

37. For DNA: 260 nm Optical Density (OD) of 1 in a 1 cm path length = 50 µg/ml - dsDNA 40 µg/ml - ssDNA and RNA Example: OD = 0.6579 0.6579 × 100 65.79 × 50 = 3289.5 µg/ml Purity of DNA: OD260 nm OD280 nm = 1.8 - 2 37

38. Thermocyclers /PCR machines Polymerase Chain Reaction (PCR) is a technique to make many copies of a specific DNA region in vitro The reaction is repeatedly cycled through a series of temperature changes, which allow many copies of the target region to be produced 38

39. 39

40. Gel Electrophoresis Apparatus Gel electrophoresis is a technique used to separate macromolecules (DNA, RNA or protein) according to their size Small pores of the matrix of the gel restrict the movements of molecules and separate them according to the size Larger molecules move slowly compared to smaller molecules The gel is composed of either Agarose or polyacrylamide 40

41. Horizontal gel electrophoresis ( Agarose ) 41

42. Vertical gel electrophoresis (Polyacrylamide) 42

43. Gel Documentation System Usage: imaging and documentation of nucleic acid and protein suspended with agarose or polyacrylamide gel. 43

44. Particle bombardment / Gene gun Usage: to deliver exogenous DNA, RNA or protein to cells Based on direct delivery of particles into plant cells using gold or tungsten particles 44

45. 45

46. Summary There are several laboratory equipment related to molecular biology Each of them has unique working principle and usages Laboratory equipment should be handled very carefully 46

47. Thank You 47