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
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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
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3. Basic laboratory equipment and consumables in
molecular biology
Equipment
1. Autoclave
2. Incubator
3. Microwave oven
4. Shaker
5. Water bath
6. Centrifuge
7. Vortex mixer
8. Thermocylers/PCR machine
9. Gel electrophoresis apparatus
10. Gel documentary system
11. Particle bombardment/Gene
gun
Consumables
1. Micropipettes
2. Glass wares
3. Nitrile gloves
4. Tubes
• Eppendorf tubes
• Centrifuge tubes
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4. 1. 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
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5. Pipette holder
Pipette tips
Pipette tips box
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6. Unit conversions
1 l = 1000 ml
1 ml = 1000 µl
1 kg = 1000 g
1 g = 1000 µg
1 ml = 1 cm3
1 m3 = 1000 l
1 ppm (parts per million) = 1 mg/l = 1 µg/ml
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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
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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
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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
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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
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11. Volumetric flask
Conical flask
Beaker
Test tube
Graduated cylinder 11
2. Glass wares….

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

13. 3. 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: 121oC
• Duration : 30 minutes
• Pressure : 1.06 kg/cm2 (15 psi)
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14. 4. Incubator
• Usage: to provide a controlled, contaminant-
free environment for cell and tissue culture
• Maintains optimal
• Temperature
• Humidity
• CO2 and O2 content
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15. 5. Microwave oven
• Usage: to heat laboratory samples and to prepare
solutions
• Uses microwaves (radio waves) to heat the
samples
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16. 6. Water bath
• Usage: to incubate samples in water at a
constant temperature over a long period of
time
• Can be used up to 99.9oC
• Shaking water bath has extra control for
shaking, which moves liquid around
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17. 7. Shaker
• Usage: to mix small volume of solutions
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18. 8. 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
(-195.79oC)
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20. 9. Vortex mixer
• Usage: mixing small vials of liquids
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21. 10. 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
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23. Rotates about
an axis resulting
in strong force
perpendicular to
the axis
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24. Types of rotors
Fixed angle rotors
Swinging bucket
rotors / horizontal
rotors
Vertical rotors
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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
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26. Types of centrifuge
1. Bench top centrifuge
• It is a compact centrifuge commonly used
in clinical and research laboratories
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27. 2. Low-speed centrifuge
• Used in laboratories for the routine separation of
particles
• Speed: 4000-5000 rpm
• Usually operated under room temperature
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28. 3. 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
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29. 4. Micro centrifuge
• Used for separation of samples with
smaller volumes (0.5 – 2 µl)
• Speed: 12000 – 13000 rpm
• Used for separation of cell organelles
like nuclei and DNA
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30. 5. Refrigerated centrifuge
• These centrifuges are provided with temperature
control ranging from -20oC to -30oC
• Have a temperature control unit
• Used to separate yeast cells, chloroplasts and
erythrocytes
• Speed at 30,000 rpm
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31. 6. 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
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32. 11. 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
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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
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34. 12. 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
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35. Types of spectrophotometer
1. UV and visible spectrometer
2. Infrared (IR) spectrometer
3. Atomic absorption spectrometer
4. Atomic emission spectrometer
5. X-ray fluorescence spectrometer
6. Mass spectrometer
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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
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38. 13. 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
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40. 14. 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
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41. Horizontal gel electrophoresis
(Agarose)
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42. Vertical gel electrophoresis
(Polyacrylamide)
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43. 15. Gel Documentation System
• Usage: imaging and documentation of nucleic acid and protein
suspended with agarose or polyacrylamide gel.
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44. 16. 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
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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
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47. Thank You
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