Dark Field Microscopy

Hello readers ^_^

Hope you all are studying well.

Today let's discuss Dark field microscopy. There are lot of different microscopes and microscopic techniques. We will try covering most and if possible all of them in coming time.

ELISA

Howdie guys, Toshlan.
What is that?
I don't know:p I guess it's a hello or welcome...in Swedish.
-_-
Let's get back to the point before we get off topic.
Today we will be learning about ELISA. You can check her at Instagram @elisaimmunomedicotechniconaruto..
Will You shut up.
Sorry, let's begin. Go.

Pulse field gel electrophoresis

Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric field that periodically changes direction.Standard gel electrophoresis techniques for separation of DNA molecules provided huge advantages for molecular biology research. However, it was unable to separate very large molecules of DNA effectively. 

DNA molecules larger than 15-20kb migrating through a gel will essentially move together in a size-independent manner.

In DNA electrophoresis by the standard method however, DNA molecules larger than 20kb show essentially the same mobility in a static electric field, making differentiation between these DNA molecules impossible.The first attempts to resolve these larger fragments included using low percentage agarose gels and low voltage gradients. Even under these extreme conditions, separation of large DNA molecules was difficult .

In 1984, David Schwartz was able to offer a new technique. He suggested that periodically changing the orientation of the electric field would force DNA molecules in the gel to relax upon the removal of the first field and elongate to align with the new field. It was his assumption that this process should be size dependent. Schwartz was finally able to demonstrate the effectiveness of this technique when he successfully separated yeast chromosomes that were several hundred kilo bases in length.

This technique became known as Pulsed Field Gel Electrophoresis (PFGE). The development of PFGE expanded the range of resolution for DNA fragments by as much as 2 orders of magnitude.

Procedure

• This method basically involves electrophoresis in agarose where two electric fields are applied alternately at different angles for defined time period (eg.6Osec)
• Activation of first electric field causes the coiled molecules to be stretched in the horizontal plane & start to move through the gel.
• Interruption of this field & application of second field force the molecule to move in the new direction .
• Since there is a length dependent relaxation behavior when long chain molecule undergo conformational change in an electrical field, the smaller a molecule , the quicker it  realigns  itself with new field and is able to moving through the gel. Larger molecules take longer to realign.
• In this way , with continual reversing of the field ,smaller molecule draw ahead and separate 

APPLICATION

Pulsed-field gel electrophoresis (PFGE) is a highly discriminative molecular typing technique that is used in epidemiological studies worldwide. 

• PFGE may be used for genotyping or genetic fingerprinting. It is commonly considered a gold standard in epidemiological studies of pathogenic organisms. 
• Subtyping has made it easier to discriminate among strains of Listeria monocytogenes and thus to link environmental or food isolates with clinical infections.
• Identifying the course of  bacterial foodborne illness(eg.Salmonella infections).
• The precise analysis provided by PFGE is unparalleled, but the process does require greater investment of funds and time. 

-Dixy

Extraction and Purification of Plasmid DNA by Alkaline Lysis Method

Hello everyone,

Today Iam here with another method for Isolation of DNA. Here I will be mainly focusing on Extraction and Purification of Plasmid DNA by Alkaline Lysis Method. 

A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes. Scientists have taken advantage of plasmids to use them as tools to clone, transfer, and manipulate genes. Plasmids that are used experimentally for these purposes are called vectors. Hence there must be a process which help us to isolate one.The method which is used for Extraction and Purification of Plasmid DNA is Alkaline Lysis Method.

Alkaline lysis is the method of choice for isolating circular plasmid DNA, or even RNA, from bacterial cells. It is probably one of the most generally useful techniques as is a fast, reliable and relatively clean way to obtain DNA from cells. If necessary, DNA from an alkaline lysis prep can be further purified.

There are basically 3 steps involved 

1) Growth of bacterial culture

The procedure starts with the growth of the bacterial cell culture (E.Coli containing PUC-18 ) harboring your plasmid. St LB Medium containing 100mcg/ml of ampicillin.

2) Harvesting and Lysis of Bacteria

When sufficient growth has been achieved, the cells are pelleted by centrifugation to remove them from the growth medium. Lysis by any one treatment , non-ionic or ionic Detergents, Organic Solvents, alkali or heat. SDS is used to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and your plasmid, to single stranded DNA (ssDNA).

3)Neutralization

Addition of potassium acetate  returns decreases the alkalinity of the mixture. Under these conditions the hydrogen bonding between the bases of the single stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA. 

5. Cleaning and concentration

Now your plasmid DNA has been separated from the majority of the cell debris but is in a solution containing lots of salt, EDTA, RNase and residual cellular proteins and debris, so it’s not much use for downstream applications. The next step is to clean up the solution and concentrate the plasmid DNA.

There are several ways to do this including phenol/chloroform extraction followed by ethanol precipitation and affinity chromotography-based methods using a support that preferentially binds to the plasmid DNA under certain conditions of salt or pH, but releases it under other conditions. 

But easiest method for purification is Agarose Gel Electrophoresis to seperate plasmid DNA .

-Dixy

Polyacrylamide Gel Electrophoresis (PAGE)

Hello everyone,

In the following section You can find some important point of Polyacrylamide Gel Electrophoresis

It is most widely used method for analyzing protein mixtures qualitatively. It is particularly useful for monitoring protein purification. As this method is based on separation of proteins according to the size it can also be used to determine the relative molecular mass of proteins. Crossed linked polyacrylamide gels are formed from the polymerization of acrylamide monomer in the presence of smaller amount of N,N methylene –bisacrylamide .

Bisacrylamide is essentially two acrylamide molecules linked by methylene group.It is used as cross linking agent.

Acrylamide monomer is polymerised in a head to tail fasion into long chains and occasionally a bis-acrylamide molecule is built into the growing chain.Thus introducing a second site for chain extension.Polymerization of acryamide  is eg. Of free radical catalysis.

FREE RADICAL CATALYSIS-

Addition of ammonium persulphte and TEMED (N,N,N’,N’-Tetramethylene diamine

S2o8 2- +e-     --------------     So4 -2 +So4-*

Free radical catalysis-free radicals are highly reactive species due to presence of unpaired electron that need to be pair with another electron to stabilize the molecule.

For eg. If free radical presented as R* and M as an acrylamide molecule, then the polymerization can be represented as follows:

R* + M ------------- RM*

RM* + M - ------------ RMM*

RMM* + M ---------------- RMMM*

Care must be used when creating this type of gel, as acrylamide is a potent neurotoxin in its liquid and powdered form.

TEMED-(N,N,N’,N’-tetra methylene diamine)-It catalyses the decomposition of persulphate ion to give free radical .( C6H16N2; MW: 116.21).TEMED stabilizes free radicals and improves polymerization. The rate of polymerization and the properties of the resulting gel depend on the concentrations of free radicals. Increasing the amount of free radicals results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. 

Decreasing the amount shows the reverse effect. The lowest catalytic concentrations that will allow polymerization in a reasonable period of time should be used. APS and TEMED are typically used at approximately equimolar concentrations in the range of 1 to 10 mM

Photopolymerisation

Photopolymerisation is an alternative method that can used to polymerize acrylamide gels.Use of riboflavin. Photodecomposition of riboflavin generates a free radical. 

With love 

-Dixy

AGAROSE GEL ELECTROPHORESIS

Hello everyone,

Today I will be writing few important points about Agarose Gel Electrophoresis,  as this the basis technique employed in Microbiological laboratory .  All the student of Microbiology should know this technique , So are You all ready to learn about Agarose Gel Electrophoresis 

The term electrophoresis  describes a   migration of charged ions under  electric field. The charged particles of various biological molecules will migrate either  to the  cathode or to the anode depending on the nature  of  their net charge. The equipment  required  for electrophoresis  consist basically of  two items  a power pack and electrophoresis unit

A power pack supplies a direct current between the electrodes in the electrophoresis unit. Electrophoresis units are available for running  either vertical or horizontal gel systems.

Agarose is a linear polyacrylamide (average relative molecular mass about 12000)made up of the basic repeat unit agarobiose,which comprises alternating units of galactose and 3,6-anhydrogalactose

Agarose gels are made by suspending dry agarose in aqueous buffer (TAE),then boiling the mixture until a clear solution forms. This is poured and allow to cool at room temperature to form a rigid gel.The gelling properties are attributed to both inter -and intramolecular hydrogen bonding  within and between the long agarose chains. This cross-linked structure gives the gel good anticonvectional properties. The pour size in the gel is controlled by the initial concentration of agarose,the large pour sizes are formed from low concentrations and smaller pour size formed from higher concentrations.

Agarose is used in gel electrophoresis to separate nucleic acids (like DNA) by size, charge and other physical properties. Gel electrophoresis uses an electrical current to make particles move. For example, DNA is negative, so it will travel towards to positive electrode of the gel box.

Agarose has small pores through which a DNA can travel. Bigger fragments of DNA travel shorter distances, because it takes longer for them to navigate through the pores of the agarose gel. Identically sized pieces of DNA will travel the same distance, which is why we get bands (DNA with loading dye) after we run a gel

PROCEDURE

1)Pour the gel slowly into the tank,leave to set for at least 30 minutes, preferably 1 hour, with the lid on then add Ethidium Bromide.

2)Pour TAE buffer into the gel tank to submerge the gel to 2–5 mm depth. This is the running buffer

3)Add an appropriate amount of loading buffer with DNA sample which will be used as sample in this electrophoresis

4)Load the first well with marker,then other wells with different samples of DNA

5)Close the gel tank, switch on the power-source and run the gel at 5 V

6)Monitor the progress of the gel by reference to the marker dye

Advantage

Relatively inexpensive, non-toxic, does not cause the sample to denature, so at the end of the electrophoresis procedure, precious or limited samples can still be recovered and used for other experimental procedures. It can give very good resolution and separation of large from small molecules as the agarose gel's pore size can be specified by the user, and only requires small amounts of sample. The process is fast (minimum of 30 minutes), and the apparatus is easy to set up and operate.

Disadvantage

If there is an increase in temperature there is chances in genetic material can adopt the shapes which are not needed.If the molecules are very similar in size, then it may be difficult or impossible to resolve or separate them from each other. Quantification of the amount of a molecule by observing the size or intensity of fluorescence of a band may be difficult.

Applications

• Separation of RNA and protein molecules
• Forensic science
• DNA fingerprinting
• Recombinant DNA technology
• Cloning
• Used in southern and northern blotting

Western Blotting

Hello Readers..

Today we learn something about Western blotting. I do not have an idea as to why they it is called western, but let's get an idea about blotting technique.

Western blot is a technique to detect specific protein present in any sample. 

Well to identify the proteins, first they need to be separated which is carried out by gel electrophoresis. You can check it out over here.

After separation of the protein fragments, they are transferred onto nitrocellulose membrane.
Nitrocellulose membrane is used because proteins bind well to it.

There can be two methods for this transfer :-
a) This is a newer method using electricity. Voltage is applied across the gel and the fragments are pulled on to the membrane and maintain their original organisation.

b) This is a traditional method in which capillary movement of the buffer is used. This method is a bit time consuming. The nitrocellulose sheet is placed above the gel , and a weight is kept at the top. the entire stack is kept in a transfer buffer. the buffer moves up due to capillary action and brings the proteins from the gel onto the membrane in the same organisation.

The next step is probing.
Antibodies specific for the target proteins are used as a probe. This will be our primary antibody. These probes will bind to the target proteins, and any unbound antibodies are washed off. Any non specific binding, like binding of the probe to the nitrocellulose sheet is avoided by blocking.

Blocking is a step done prior to addition of the probe. they membrane is immersed into a solution of Bovine Serum Album (Rich source of Protein) in buffered Saline. the proteins in BSA bind to the nitrocellulose membrane all over where the target proteins have not bound (as in they fill up all the empty space on the membrane), thus leaving no room for the probes to bind.

After incubation post probing for 24 hrs at 4oC, a secondary antibody which will be specific to the primary antibody is added to sort of amplify the signal. Our this secondary antibody, like in ELISA will be something that produces signal like production of a colored reaction or radioactivity. Reporter enzymes are widely used for production of luminiscence. After addition of secondary antibody, the membrane is incubated for 2-4 hours at room temperature.

Next step is detection:
Detection of the target protein will vary according to the secondary probe used.
For eg.
For a colored substrate a colorimetric detection will be used.

For a radioactive label radioactivity detection is required. this can be done simply by exposing a X ray film to the membrane and it will develop showing darker spots where the label must have bound the target protein, etc.

Western blotting is a very important technique in practicals as well as exam point in view.
Hope this post helps you all learn.
Keep learning and do not forget to be amazing <3

Lots of love
-Staph.

Indirect ELISA

Enzyme-linked immunosorbent assay, commonly known as ELISA (or EIA), is similar in principle to RIA but depends on an enzyme rather than a radioactive label. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate.

INDIRECT ELISA

Here, in Indirect ELISA steps are slightly different, though the basic principle remains the same.

Two different antibodies are used.

1. Primary antibody, let's consider this as Ab 1.
2. And a Secondary antibody, Ab 2.

A serum sample or any other sample containing the primary antibody is added to a microtitre plate containing the antigens.

Reaction is allowed between them. and any free Ab 1 is washed off.

Then a secondary antibody(Anti-isotype antibody) linked to an enzyme, specific to the primary antibody is added

Reaction is allowed between them. and any free Ab 2 is washed off.

A substrate specific to the enzyme is added and a chromogenic reactions occurs between the two.

This can be measured by spectrophotometery.

Uses: Generally, serum antibodies to HIV can be detected by indirect ELISA within 6 weeks of infection. 

That's all

Hope it helps

Lots of Love and be Amazing!!

-Staph

Isolation of DNA

Hello everyone ,

DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher. Currently it is a routine procedure in molecular biology or forensic analyses.Purification or isolation of nucleic acids is the first step in most molecular biology studies and all recombinant  DNA techniques. Today I will be writing about Isolation of DNA from cell .

Source : the simplest source of DNA could be bacteria

The methods include

1) Cell lysis: It solubilizes Nucleic acid . This step include 

                      addition of SDS /Alkali

                       Boiling

                       addition of Chaotropic agents

2) Enzymatic treatment:Addition of protease (Protinase K), inhibit DNAse activity

                                      Addition of Nucleases, removes unwanted nucleic acid (RNAse or DNAse)

3) Phenol extraction:  Here equal volume volume of phenol is added to the sample and centrifuged. This allows partition of phase . As proteins are hydrophobic, thus partition in organic phase. Also DNA seen in aqueous phase in upper layer .

4) Ethanol precipitation: Here 0.3M sodium Acetate/ Potassium acetate / Ammonium acetate( pH 5-5.5) is added to neutralize the highly charged backbone and promote hydrophobic interactions.then equal volume of Ethanol /Isopropanol is added and incubated overnight at 4 °C .the precipitated DNA is collected by centrifugation . The pellet is rinsed with 70% Ethanol to remove excess of salt, dried and dissolved in buffer .

5)Adsorption Methods: This step mainly separates RNA from sample.

The above sample as obtained above is mixed with a resin ( silica or diatomaceous earth). the DNA binds to the matrix but RNA do not. the DNA is eluted with low salt buffer.

ThankYou

with love 

-Dixy

Paper Chromatography.

Hello everyone..

Today I am talking about PAPER CHROMATOGRAPHY..

So Paper...because we are using Whottman's filter papers here.

Chrome means color, graph essentially means separating. Hence the simple definition is separation of components in the mixture, which are later detected by a color developing reagent that is specific for a particular component.

I am still researching as to what ATO means, in case you find out before me, then please do inform :p

You can also use the technique for identification of the components in the mixture, by plotting the standard samples in the same paper. 

The sample mixture is applied to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action.Components of the mixture are carried along with the solvent up the paper to varying degrees, depending on the compound's preference to be adsorbed onto the paper versus being carried along with the solvent. 

The paper is composed of cellulose to which polar water molecules are adsorbed, while the solvent is less polar, usually consisting of a mixture of water and an organic liquid. The paper is called the stationary phase while the solvent is referred to as the mobile phase. 

Performing a chromatographic experiment is basically a three-step process: 

1) application of the sample, 

2) "developing" the chromatogram by allowing the mobile phase to move up the paper, and 

3) calculating Rf values and making conclusions.

Solvent systems and the color developing reagents differ on the basis of the samples that is being seperated.
For eg: In detection of amino acids, the most common solvent system used is - Butanol:acetic acid:water in proportions of 40:10:10.
Spraying reagent used is ninhydrin.

A modified technique is to add the color developing reagent in to the solvent system. this avoids the later need to dry, spray and incubate the paper.


In order to obtain a measure of the extent of movement of a component in a paper chromatography experiment, we can calculate an "Rf value" for each separated component in the developed chromatogram. An Rf value is a number that is defined as:

Rf - Distance traveled by component from application point.(A)
       ____________________________________________
       Distance traveled by solvent from application point (B)

Hope it helps you all.

Thank you.

Lots of love, don't forget to be amazing.

-Staph.

Radioimmunoassay (RIA)

Important points to remember (And to impress the examiner :p )

1. First developed in 1960 by two endocrinologists, S. A. Berson and Rosalyn Yalow, to determine levels of insulin–anti-insulin complexes in diabetics.
2. Sensitive technique - measures hormones, serum proteins, drugs, and vitamins at concentrations of 0.001 micrograms per milliliter or less.
3. Antigen is generally labeled with a gamma-emitting isotope such as 125 I, but beta-emitting isotopes such as tritium (3 H) are also routinely used as labels.

Principle: 

RIA involves competitive labeling of the labeled and the unlabeled antigen to the antibody.

Labeled antigen at a saturation concentration (don't worry,it means concentration at which all the binding sites of antibodies are occupied)  + Antibody

Then test samples of unlabeled antigen of unknown

concentration are added in  larger 

amounts.

Now here, the antibody is unable to recognize the difference between the labeled and the unlabeled antigen,so 

the two kinds of antigen compete for available binding sites on the antibody.

As the concentration of the unlabeled antigen increases, the antibody just like a betraying friend or any douchebag boyfriend unbinds the already labeled antibody, and like a snatchy girlfriend the unlabeled antigen displaces the labeled one and occupies the site.

The decrease in the amount of radiolabeled antigen bound to specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the test sample.

To determine the amount of labeled antigen bound, the Ag-Ab complex is precipitated to separate it

from free antigen (antigen not bound to Ab), and the radioactivity in the precipitate is measured. 

A standard curve can be generated using unlabeled antigen samples of known concentration (as a standard sample), and from this plot the amount of antigen in the test mixture may be precisely determined.

Thanks you.

Lots of Love <3

-Staph.

Sodium Doedecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE)

Hello everyone , 

Dixy is back with new experience to be shared  :)

Today I will be writing few important points about SDS PAGE Electrophoresis,  as I have used this technique many time during my Post graduation  . 

When I first performed SDS , that time I didn't knew  even to seal the apparatus , but now i can interdependently use it.  So don't be afraid of this technique , you can also do it easily by just following few points which I will be mentioning below.

 You must have seen the apparatus

 It consists of 2 plates, a comb,2 clamps, 2 side spacer, a buffer tank , power pack

Do you know you can prepare gel of different porosity by mixing  ingredients in different concentration 

• But the first thing is to do is sealing the apparatus with 1 % Agarose.
• All the step should be carried out with Gloved hands.
• All ingredient should be prepared fresh.
• Also point should be noted , to mix the ingredients gently , ensuring no air bubbles forms.
• Carefully pour the mixture of gel directly into glass plate assembly as soon the TEMED is added ,  not forgetting the fact that the mixture contains acrylamide and TEMED which are carcinogenic .
• Add water above the Gel mixture which allows polymerization of gel
• Once  comb is placed , don't move it , this way u will not get uniform wells.
• Fill the buffer in buffer tank 
• Always add sample in small volume with micropipette
• Cover the lid and connect the electrophoresis tank to the power supply.
• Allow the sample to run till it reaches 3/4 of gel
• while separating  gel  from plate , care should be taken not to tear the gel, or else u may lose separated  proteins.
• The gel is stained with Coomassie blue stain or Silver stain ,and destained 

Hope this may help you,

Thank you

With love ,

-Dixy