Fluorescence Microscope

FLUORESCENCE MICROSCOPE

 
A fluorescence microscope is basically a conventional light microscope with added features and components that extend its capabilities.

·         A conventional microscope uses light to illuminate the sample and produce a magnified image of the sample.

·         A fluorescence microscope uses a much higher intensity light to illuminate the sample. This light excites fluorescence species in the sample, which then emit light of a longer wavelength. A fluorescent microscope also produces a magnified image of the sample, but the image is based on the second light source -- the light emanating from the fluorescent species -- rather than from the light originally used to illuminate, and excite, the sample.

Working Principle:

In most cases the sample of interest is labelled with a fluorescent substance known as a fluorophore and then illuminated through the lens with the higher energy source. The illumination light is absorbed by the fluorophores (now attached to the sample) and causes them to emit a longer lower energy wavelength light. This fluorescent light can be separated from the surrounding radiation with filters designed for that specific wavelength allowing the viewer to see only that which is fluorescing.

The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light from the image. First, the microscope has a filter that only lets through radiation with the specific wavelength that matches your fluorescing material. The radiation collides with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light. To become detectable (visible to the human eye) the fluorescence emitted from the sample is separated from the much brighter excitation light in a second filter. This works because the emitted light is of lower energy and has a longer wavelength than the light that is used for illumination.

Most of the fluorescence microscopes used in biology today are epi-fluorescence microscopes, meaning that both the excitation and the observation of the fluorescence occur above the sample. Most use a Xenon or Mercury arc-discharge lamp for the more intense light source.

Instrumentation:

Nearly all fluorescence microscopes have following basic parts below.

·         Focus Illumination

·         Collector for emitted fluorescence

·         Dichroic mirror

·         Filter:

1.       Focus the illumination (excitation) light on the sample.

In order to excite fluorescent species in a sample, the optics of a fluorescent microscope must focus the illumination (excitation) light on the sample to a greater extent than is achieved using the simple condenser lens system found in the illumination light path of a conventional microscope.

2.       Collect the emitted fluorescence:

This type of excitation-emission configuration, in which both the excitation and emission light travel through the objective, is called epifluorescence. The key to the optics in an epifluorescence microscope is the separation of the illumination (excitation) light from the fluorescence emission emanating from the sample. In order to obtain either an image of the emission without excessive background illumination, or a measurement of the fluorescence emission without background "noise", the optical elements used to separate these two light components must be very efficient.

3.      The Dichroic Mirror

In a fluorescence microscope, a dichroic mirror is used to separate the excitation and emission light paths. Within the objective, the excitation emission share the same optics.

The excitation light reflects off the surface of the dichroic mirror into the objective.

The fluorescence emission passes through the dichroic to the eyepiece or detection system.

The dichroic mirror's special reflective properties allow it to separate the two light paths. Each dichroic mirror has a set wavelength value -- called the transition wavelength value -- which is the wavelength of 50% transmission. The mirror reflects wavelengths of light below the transition wavelength value and transmits wavelengths above this value. This property accounts for the name given to this mirror (dichroic, two color). Ideally, the wavelength of the dichroic mirror is chosen to be between the wavelengths used for excitation and emission.

The dichroic mirror is a key element of the fluorescence microscope, but it is not able to perform all of the required optical functions on its own. Typically, about 90% of the light at wavelengths below the transition wavelength value are reflected and about 90% of the light at wavelengths above this value are transmitted by the dichroic mirror. When the excitation light illuminates the sample, a small amount of excitation light is reflected off the optical elements within the objective and some excitation light is scattered back into the objective by the sample. Some of this "excitation" light is transmitted through the dichroic mirror along with the longer wavelength light emitted by the sample. This "contaminating" light would otherwise reach the detection system if it were not for another wavelength selective element in the fluorescence microscope: an emission filter.

Figure 1: Optical diagram of Fluorescent Microscope

4.      Excitation and Emission Filters

Two filters are used along with the dichroic mirror:

Excitation filter -- In order to select the excitation wavelength, an excitation filter is placed in the excitation path just prior to the dichroic mirror.

Emission filter -- In order to more specifically select the emission wavelength of the light emitted from the sample and to remove traces of excitation light, an emission filter is placed beneath the dichroic mirror. In this position, the filter functions to both select the emission wavelength and to eliminate any trace of the wavelengths used for excitation.

These filters are usually a special type of filter referred to as an interference filter, because of the way in which it blocks the out of band transmission. Interference filters exhibit an extremely low transmission outside of their characteristic bandpass. Thus, they are very efficient in selecting the desired excitation and emission wavelengths.

Applications:

The refinement of epi-fluorescent microscopes and advent of more powerful focused light sources, such as lasers, has led to more technically advanced scopes such as the confocal laser scanning microscopes and total internal reflection fluorescence microscopes (TIRF).

CLSM's are invaluable tools for producing high resolution 3-D images of sub-surfaces in specimens such as microbes. Their advantage is that they are able to produce sharp images of thick samples at various depths by taking images point by point and reconstructing them with a computer rather than viewing whole images through an eyepiece.

These microscopes are often used for -

·         Imaging structural components of small specimens, such as cells

·         Conducting viability studies on cell populations (are they alive or dead?)

·         Imaging the genetic material within a cell (DNA and RNA)

·         Viewing specific cells within a larger population with techniques such as FISH

Microarrays

MICROARRAY

INTRODUCTION:

The term microarray was first introduced by Schena et al. in 1995 and the first genome of an eukaryotic species completely investigated (Saccharomyces cerevisiae) by a microarray was published in 1997 (Lashkari et al., 1997). In the last few years, further improvements were made especially when substituting the immobilized DNA-probes derived from clone-libraries by chemically synthesized oligonucleotides.

There are different names for the microarrays, like DNA/RNA Chips, BioChips or GeneChips. The array can be defined as an ordered collection of microspots, each spot containing a single defined species of a nucleic acid. The microarray technique is based on hybridization of nucleic acids. In this technique, sequence complementarity leads to the hybridization between two single-stranded nucleic acid molecules, one of which is immobilized on a matrix. There exist two variants of the chips: cDNA microarrays and oligonucleotide arrays. Although both the DNA and oligonucleotide chips can be used to analyze patterns of gene expression, fundamental differences exist between these methods. Two commonly used types of chips differ in the size of the arrayed nucleic acids. In cDNA microarrays, relatively long DNA molecules are immobilized by high-speed robots on a solid surface such as membranes, glass or silicon chips. Sample DNAs are amplified by the polymerase chain reaction (PCR) and usually are longer than 100 nucleotides. This type of arrays is used mostly for large-scale screening and expression studies.

The oligonucleotide arrays are fabricated either by in situ light-directed chemical synthesis or by conventional synthesis followed by immobilization on a glass substrate. Those with short nucleic acids (oligonucleotides up to 25 nucleotides) are useful for the detection of mutations and expression monitoring, gene discovery and mapping. In the procedure of genomic analysis, both types of microarrays are exposed to a labelled sample, hybridized, and complementary sequences are determined.

PRINCIPLE:

mRNA is an intermediary molecule which carries the genetic information from the cell nucleus to the cytoplasm for protein synthesis. Whenever some genes are expressed or are in their active state, many copies of mRNA corresponding to the particular genes are produced by a process called transcription. These mRNAs synthesize the corresponding protein by translation. So, indirectly by assessing the various mRNAs, we can assess the genetic information or the gene expression. This helps in the understanding of various processes behind every altered genetic expression. Thus, mRNA acts as a surrogate marker. Since mRNA is degraded easily, it is necessary to convert it into a more stable cDNA form. Labeling of cDNA is done by fluorochrome dyes Cy3 (green) and Cy5 (red). The principle behind microarrays is that complementary sequences will bind to each other.

The unknown DNA molecules are cut into fragments by restriction endonucleases; fluorescent markers are attached to these DNA fragments. These are then allowed to react with probes of the DNA chip. Then the target DNA fragments along with complementary sequences bind to the DNA probes. The remaining DNA fragments are washed away. The target DNA pieces can be identified by their fluorescence emission by passing a laser beam. A computer is used to record the pattern of fluorescence emission and DNA identification. This technique of employing DNA chips is very rapid, besides being sensitive and specific for the identification of several DNA fragments simultaneously.

Figure1: Principle of Microarray with reference to mRNA chip

PROCEDURE:

1.      Sample Collections: The samples can be a variety of organism. E.g. Two samples: cancerous human skin tissue and healthy human skin tissue

2.      Isolation of Nucleic acid: Every microarray study starts with the isolation of the respective targets (e.g. DNA or RNA). In principle, nucleic acids are isolated upon cell disruption by mechanical or enzymatic methods and precipitated at increased salt concentrations or ethanol.

mRNA Isolation: The extracted of RNAs using any methods (phenol-chloroform) is passed through the column containing beads with poly-T tails which bind the mRNA as it has a poly-A tail. All tRNA and rRNA are wshed out in this technique. Then it is rinsed with buffer to release the mRNA by disrupting the hybrid bonds with pH disturbance.

3.      Labelling: The next step is labelling of the molecules with fluorescent dyes; for that step various methods exist. Often the labelling step is done during the enzymatic amplification reaction at which fluorescently labelled nucleotides or primers are incorporated into the newly synthesized amplicons. Nowadays a broad range of fluorescent dyes with different absorption and excitation wavelengths are available. The different absorption and excitation maxima allow the combination of fluorescent dyes. In microarray analyses the fluorescent dyes Cy3 and Cy5 are widely used. cDNA labelled: The labelling mix contains poly-T primers, reverse transcriptase (to make cDNA) and fluorescently dyes nucleotides. The cyanine 3 (cy3-flouresce green) to the healthy cells and cyanine 5 (cy-5-fluoresce red) to the cancerous cells in this experiment.

4.      After purification of the labelled amplicons, these molecules are mixed with a hybridization buffer and are subsequently applied to the microarrays coating ssDNA probes and incubated overnight. After the hybridization procedure, unbound molecules have to be washed off before the detection can be done by laser-scanning with dye specific wavelengths.

5.      The detection step generates an image of the microarray, which is employed for raw data extraction. Thus, fluorescent intensities of each single spot of the microarrays are measured and written in a results file along with the spot coordinates and the specific “gene” identifier. The intensity of the generated signal depends on the number of molecules (targets), which have bound to the probe-molecules within one spot (also called feature). E.g. the higher intensity for red color indicates the up regulation for cancer cells and green intensities indicate the down regulation of gene expression for cancer cells.

6.      The last step in a microarray experiment is the bioinformatic analysis of the data of a single slide or data from many samples of distinct classes processed in parallel within one experiment.

Figure 2: The methods of microarrays

APPLICATIONS

·         The GeneChip technology may be employed in diagnostics (mutation detection), gene discovery, gene expression and mapping. It is used to measure expression levels of genes in bacteria, plant, yeast, animal and human samples.

·         At the present time, the main large-scale application of microarrays is comparative expression analysis.  The microarray technology provides the possibility to analyze the expression profiles for thousands of genes in parallel. Another application is the analysis of DNA variation on a genome-wide scale. Both of these applications have many common requirements. By hybridization with labelled mRNA, cDNA, arrayed PCR products or oligonucleotides on a substrate have been successfully used for monitoring transcript levels, single nucleotide polymorphism (SNP), or genomic variations between different strains.

·         One of the most significant applications of this technique is, as mentioned above, gene expression profiling on the whole genomic scale. For example, the expression levels of the genes in the Saccharomyces cerevisiae genome have been successfully determined with both the DNA and oligonucleotide microarray technology.

·         This technique has also been used to investigate physiological changes in human cells. DNA microarray technology was applied to detect differential transcription profiles of a subset of the Escherichia coli genome.

·         The microarray technology is a powerful yet economical tool for characterizing gene expression, regulation and will prove to be useful for strain improvement and bioprocess development. It may prove to be useful for strain development, process diagnosis, and process monitoring in bioreactors.

·         Information obtained from DNA chip analysis may enable researchers to determine the impact of a drug on a cell or group of cells, and consequently to determine the drug’s efficacy or toxicity. Knowledge of gene expression profiles can also help researchers to identify new drug targets.

·         The BioChip opens a new world of diagnostics based on genetics. This technology may be adequate to answer many medical questions. For example, gene expression profiles can be used for classification of tumors and for prognosis.

·         The technology finds increasing application in fundamental and applied research. The  major feature of this technique is that it allows one to perform a simultaneous analysis of a great number of DNA sequences.

·         The GeneChip technology is a new technique that undoubtedly will substantially increase the speed of molecular biology research.

Clonning

CLONING

1. Cell-based DNA cloning
2. Polymerase mediated in vitro DNA cloning

The importance of DNA cloning:

Current DNA technology is based on two different approaches:

a.   Specific amplification (DNA cloning) which involves cell-based DNA cloning (involving a vector/replicon and a suitable host cell) and in vitro DNA cloning (PCR)

b.   Molecular hybridization where the DNA fragment of interest is specifically detected using a mixture of different sequences

                      

Figure 1: DNA Clonning in vivo and In Vitro

Four steps in cell-based cloning:

1. Construction of recombinant DNA molecules. Involves the use of endonuclease restriction enzymes, ligation, and a replicon (vector).
2. Transformation in appropriate host cells.
3. Selective propagation of cell clones. This step takes advantage of selectable markers.
4. Isolation of recombinant DNA from cell clones followed by molecular characterization (such as restriction enzyme analysis).

Recombinant DNA Libraries (3 types):

1. Genomic library, Collection of cloned restriction enzyme digested DNAs containing at least one copy of every DNA sequence in a genome.
2. Chromosome library, Collection of cloned restriction enzyme digested fragments from individual chromosomes.
3. Complementary DNA (cDNA) library, Collection of clones of DNA copies made from mRNA isolated from cells.

·         reverse transcriptase (RNA dependent DNA polymerase)

·         Synthesizes DNA from an RNA template

·         cDNA libraries reflect what is being expressed in cells.

1. Genomic Library:

3 ways to make a genomic library:          

1. Complete digestion (at all relevant restriction sites)

1. Produces a large number of short DNA clones.
2. Genes containing two or more restriction sites may be cloned in two or more pieces.

2. Mechanical shearing

1. Produces longer DNA fragments.
2. Ends are not uniform, requires enzymatic modification before fragments can be inserted into a cloning vector.

3.      Partial digestion

1. Cut at a less frequent restriction site and limit the amount and time the enzyme is active.
2. Results in population of large overlapping fragments.
3. Fragments can be size selected by agarose electrophoresis.
4. Fragments have sticky ends and can be cloned directly.

Screening a genomic library (plasmid or cosmid):

1.      Plasmid vectors containing digested genomic DNA are transformed into E. coli and plated on selective medium (e.g., ampicillin).

2.      Colonies that grow are then are replicated onto a membrane (E. coli continues to grow on the membrane).

3.      Bacteria are lysed and DNA is denatured.

4.      Membrane bound DNA is next probed with complementary DNA (e.g., 32P radio-labeled DNA).

5.      Complementary DNA in the probe is composed of DNA sequence you are looking for; homologous sequence presumably also found in library.

6.      Unbound probe DNA is washed off the filter. 

7.      Hybridization of probed DNA is detected by exposure to X-ray film (or by chemiluminescence).

8.      Pattern is noted from exposure pattern of clones on X-ray film.

9.      Select clones that test positive and isolate for further analysis.

2. Chromosome Library:

1. Screening can be reduced if target genes can be localized to a particular chromosome.
2. Chromosomes can be separated by flow cytometry.

                                I.      Condensed chromosomes are stained with fluorescent dye.

                             II.      Chromosomes separate based on the level of binding of the dye and are detected with a laser.

3. cDNA Library:

1. cDNA is derived from mature mRNA, does not include introns.
2. cDNA may contain less information than the coding region.
3. cDNA library reflects gene activity of a cell at the time mRNAs are isolated (varies from tissue to tissue and with time).
4. mRNA degrades quickly after cell death, and typically requires immediate isolation (cryoprotectants can increase yield if immediate freezing is complicated by field work).

Creating a cDNA library:

Step 1-Isolate mRNA:

•         Mature eukaryote mRNA has a poly-A tail at the 3’ end.

•         mRNA is isolated by passing cell lysate over a poly-T column composed of oligo dTs (deoxythymidylic acid).

•         Poly-A tails stick to the oligo dTs and mRNAs are retained, all other molecules pass through the column.

Step 2-cDNA synthesis:

Step 3-Clone cDNA

•         Anneal a short oligo dT (TTTTTT) primer to the poly-A tail.

•         Primer is extended by reverse transcriptase 5’ to 3’ creating a mRNA-DNA hybrid.

•         mRNA is next degraded by Rnase H, but leaving small RNA fragments intact to be used as primers.

•         DNA polymerase I synthesizes new DNA 5’ to 3’ and removes the RNA primers.

•         DNA ligase connects the DNA fragments.

•         Result is a double-stranded cDNA copy of the mRNA.

Screening a cDNA library:

1. cDNA libraries are most often used to detect genes for proteins (cDNAs are generated for genes that are transcribed!).
2. If you know the DNA sequence for the protein coding gene you want to find, a homologous DNA probe can be used.
3. If no homologous DNA sequence is available, cDNA can be probed with an antibody that recognizes the protein.
4. Expression vector: cloned cDNA is inserted between a promoter and transcription terminator before it is transformed.
5. mRNA is transcribed from the cDNA and translated.
6. Colonies (now expressing proteins) are transferred to membrane.
7. Membrane is incubated with radioactive labeled antibody probe that recognizes the protein (non-radioactive chemiluminescent probes also are available).
8. Colonies with bound antibodies leave a dark spot on X-ray film.

                                      

  Figure 2: Methods of cDNA Clonning

Polymerase mediated in vitro DNA cloning:

Polymerase: DNA polymerase duplicates DNA. Before a cell divides, its DNA must be duplicated

Chain Reaction: The product of a reaction is used to amplify the same reaction. Thus, PCR results in rapid increase in the product.

PCR-Polymerase Chain Reaction is used to amplify a short, well-defined part of a DNA strand which is usually up to 10 kb. PCR, as currently practiced, requires several basic components such as:

—  DNA Polymerase, for amplifying DNA in vitro

—  DNA template, which contains the region of the DNA fragment to be amplified

—  Two primers, which determine the beginning and end of the region to be amplified DNA-Polymerase, which copies the region to be amplified

—  Nucleotides, from which the DNA-Polymerase builds the new DNA (dNTPs)

—  Buffer, which provides a suitable chemical environment for the DNA-Polymerase

—  Thin wall tubes and Thermal Cycler.

History:

—  Concept forward by H. G. Khorana et al. in 1971 ( before gene sequencing or a viable thermostable DNA polymerase)

—  15 years later Kary Mullis, coined PCR, and put into practice (Nobel Prize in Chemistry, October 1993)

—  In vitro DNA replication by DNA polymerase.

—  Mullis's original PCR process was very inefficient since it required a great deal of time, vast amounts of DNA-Polymerase, and continual attention throughout the PCR process.

—  Thermus aquaticus (Taq polymerase)

—  A disadvantage of Taq - sometimes makes mistakes (mutations) in the DNA sequence (no proofreading exonuclease activity)

—  Polymerases such as Pwo or Pfu, obtained from Archaea, have proofreading mechanisms

—  Combinations of both Taq and Pfu are available nowadays that provide both high fidelity and accurate amplification of DNA.

In vivo DNA replication requires many components such as DNA polymerase, DNA ligase, Primase, Helicase, Topoisomerase, Single strand binding protein etc for unwinding and synthesis of new copies of DNA. But in artificial DNA amplification, it is not possible to include all these enzymes because of their stability and regulation.

The PCR process consists of a series of twenty to thirty cycles. Each cycle consists of three steps;

      1. Denaturation

      2. Annealing

      3. Extension

Figure 3: Temperature graph for PCR 

1. Denaturation:

DNA denaturation is defined as the separation of double stranded DNA into two single stranded ones. In the PCR, Template DNA which is ds DNA (linear or circular) or cDNA (complementary DNA produced from produced mRNA by reverse transcriptase) is denatured by heating at 94-96°C. The heating in machine breaks apart the hydrogen bonds and separates apart. It should be remembered that an extended time of heating is required to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Usually, 1-2 minutes heating is required for complete denaturation

 Figure 4: DNA Denaturation by heating at  above 90oC

2. Annealing

It is a primer binding step. Primers can attach themselves to the single DNA strands. Usually 5°C below their melting temperature (45-60°C) is required for annealing of the primers. A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. The annealing time is usually 1-2 minutes.

Primers are the pairs of oligonucleotides each 18-25 nucleotides long. It should have 40%-60% GC content. The melting temperature of both should not differ by 5^oC. The 3’ terminal sequences of any primer should not be to any sequences of the other primer in the pair. The self-complimentary sequences (inverted repeats) of 3 bp is avoided in the primer. Two primers; forward primer and backward primer are used in the PCR.

What is the annealing temperature for the following primer (a 21 mer)?

                              3’-AAGCTTGTCCAGAATTTCGGC-5’

Solution: Every A and T nucleotide is responsible for 2 °C while G and C for 4 °C

      Therefore         (A+T) = 11 x 2 = 22

                              (C+G) = 10 x 4 = 40

      Now;

                                          22 + 40 = 62 °C (Melting temperature)

The annealing temperature is chosen as a few degrees below that number, so it is at about 57°C.

3. Elongation

It starts at the annealed primer and works its way along the DNA strand. The elongation temperature depends on the DNA-Polymerase. The time for this step depends both on the DNA-Polymerase itself and on the length of the DNA fragment to be amplified.

—  As a rule-of-thumb, 1 minute per 1000bp.

—  Elongation. Heat at 72°C for 45 seconds.

The DNA polymerase duplicates DNA in vivo which is necessary for reproduction of new cells. DNA strands are anti-parallel; One strand goes in 5' to 3' while the complementary strand is opposite (3' to 5' and dNTP follow standard base pairing rule). The DNA polymerase always moves in one direction (from 5’ à 3’) and incorporates the four nucleotides (A, T, G, C) to the growing chain. More than one DNA polymerases exist in different organisms. DNA polymerase needs Mg⁺⁺ as cofactor. Each DNA polymerase works best under optimal temperature, pH and salt concentration. During PCR, PCR buffer provides optimal pH and salt condition.

Taq DNA polymerase:

It is derived from Thermus aquaticus, a bacterium from hot stream. It is highly heat stable DNA polymerase which has an ideal temperature of 72°C. However the regular Taq DNA polymerase lacks 3’ - 5’ exonuclease activity needed to provide proof-reading function. Nowadays the Taq polymerase is available with proof reading function. The required temperature during DNA denaturation, annealing and extension is provided by PCR machine.

4. Repetition

Steps 2-4 are repeated 25-35 times, but with good primers and fresh polymerase, 15 to 20 cycles is sufficient. After complete cycles, the DNA of interest will be amplified to sufficient amount.  The machine itself is also used for storage of PCR products for certain period.

Applications:

—  Molecular biological technique for amplifying DNA without using a living organism, such as E. coli or yeast.

—  PCR is commonly used in medical and biological research labs for a variety of tasks, such as: 

—  Detection of hereditary diseases,

—  Identification of genetic fingerprints,

—  Diagnosis of infectious diseases,

—  Cloning of genes,

—  Paternity testing

Limitations:

The PCR technique has two limitations:

      1. Short sizes of amplified products (<5 b="" kb="">

      This is solved by doing Long-range PCR (up to tens of Kb long) which uses a mixture of two heat stable polymerases that provide optimal levels of DNA synthesis as well as a 3’ -> 5’ exonuclease activity.

     

      2. Low yields of amplifications

      Which is resolved by cloning the PCR amplified DNA fragment in a vector then propagating the vector in a cell based system (clone by A/T cloning or by using anchored PCR primers).

Advantages: Three major advantages of PCR are;

            1. Rapid

            (possible to amplify DNA from very quickly 2-3 hours than in cell based technique)

            2. Sensitive

            (possible to amplify DNA from a very small amount=pico mole of DNA)

            3. Robust

            (possible to amplify DNA from damaged tissues or degraded DNA)

Figure 5: Steps in DNA amplification by PCR

Notice for M.Sc. Admission

Coming up next "A Complete Note on Instrumentation"

A Complete Note on Instrumentation (Analytical Biochemistry)

UPENDRA THAPA SHRESTHA

A Complete Note on Instrumentation (Analytical Biochemistry)

First Edition, November-2016

© Author (Shrestha U.T.)

UPENDRA THAPA SHRESTHA, M.Sc. Microbiology (TU)

Head of Microbiology Department:  Kantipur College of Medical Science, Sitapaila, KTM

(B.Sc. and M.Sc. Microbiology affiliated to TU; CMLT and D-Pharmacy affiliated to CTEVT)

Deputy Director: Research Laboratory for Biotechnology and Biochemistry (RLABB)

(Research Scientist)

Faculty: Khwopa College (B.Sc. and M.Sc. Environment Science), Dekocha, Bhaktapur

PGT: Sainik Awasiya Mahavidhyalaya, Sallaghari, Bhaktapur

Email: upendrats@gmail.com

Domain: www.upendrats.blogspot.com

Publisher:

All rights reserved.

No part of this book may be reproduced in any form, by photocopy, microfilm, scanning or any other means, or incorporated into any information retrieval system, electronic or mechanical, without the written permission of the author.

Thank you!

UNIT-I

                                                                                       

·        1.1 Physiological Solutions

·        1.2 Cell Disruption

o   Mechanical Cell Disruption

o   Non-mechanical Cell Disruption

§  Physical Methods

§  Chemical Methods

§  Biological Methods

UNIT-II

                                                                                       

·        Protein Purification

o   Background knowledge about protein

o   Protein Isolation/Precipitation

o   Protein Purification

o   Protein Determination

o   Protein Assay

UNIT-III

                                                                                       

·        Centrifugation

o   Principle of Centrifugation

o   Centrifuges and its types

o   Differential Centrifugation

o   Density Gradient Centrifugation

UNIT-IV

                                                                                       

·        Chromatography

o   Paper Chromatography

o   Thin Layer Chromatography

o   Column Chromatography

§  Size Exclusion Chromatography

§  Ion Exchange Chromatography

§  Affinity Chromatography

§  Gas Chromatography

§  High Performance Liquid Chromatography (HPLC)

UNIT-V

                                                                                       

·        Electrophoresis

o   Principle of Electrophoresis

o   Media used in Electrophoresis

o   Agarose Gel Electrophoresis

o   Isoelectric Focus Gel Electrophoresis

o   SDS-PAGE

o   2D Gel Electrophoresis

o   Western Blotting

UNIT-VI

                                                                                       

·        Spectroscopy

o   UV-Visible Spectrophotometry

§  Colorimeter/Photometer/Spectrophotometer

o   Spectroflouremetry

o   Molecular Luminescence Spectroscopy

o   Atomic Spectroscopy

§  Atomic Absorption Spectroscopy

§  Atomic Emission Spectroscopy

§  Atomic Florescent Spectroscopy

o   Mass Spectroscopy

o   Nuclear Magnetic Resonance

UNIT-VII

                                                                                       

·        Microscopy

o   Electron Microscopy

§  Transmission Electron Microscopy

§  Scanning Electron Microscopy

o   Phase Contrast Microscopy

o   Fluorescent Microscopy

o   Gel-Doc System

UNIT-VIII

                                                                                       

·        Polymerase Chain Reaction

·        Microarrays