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Saturday, June 29, 2019

Gel Extraction : Principle, Procedure & Tips for Improved Yield

What is Gel Extraction?

Gel Extraction or Gel Isolation is a technique used to excise and extract out the desired DNA fragment of interest after the agarose gel electrophoresis.

Why is it done?

Gel Extraction is used to purify the desired DNA fragment from unwanted materials, generally used to purify Restriction Digests, PCR Amplicons, etc.


Procedure:

Following steps are involved in the Gel Extraction:

  • Run DNA on Agarose Gel Electrophoresis, Excise the desired band after electrophoresis run.
  • Dissolve the DNA containing excised gel
  • Bind DNA to Silica Column
  • Wash the Bound DNA
  • Elute the DNA from column


Principle of Gel Extraction / Nucleic Acid Purification

Most of the commercially available kits uses spin columns for efficiently purify the DNA from agarose gel or from the aqueous solutions. Upto 10ug of the DNA can be easily purified with considerable recovery using the spin columns.

Guanidine HCL or Guanidine Isothiocyante is the widely used chaotropic salt.The adsorption of DNA to the spin column membrane occurs at higher chaotropic salt concentration, as the salt modifies the structure of water.

Adsorption of DNA to the membrane is also depended on pH. 95% adsorption at pH <7 .5.="" before="" column="" if="" is="" loading="" of="" ph="" solution="" spin="" the="" to="">7.5, a small amount of 3M sodium acetate pH 5.0 can be added to bring the pH below 7.5.

pH dependence of DNA adsorption to Spin Column membranes.

Elution:

Elution efficiency is depended on the salt concentration and the pH of the elution buffer. Elution efficiency is higher under basic condition and at low salt concentration. Low salt elution buffer 10mM Tris.Cl or Water can be used for elution. While using water for elution, pH should be with in 7.0 - 8.5.


DNA yield & Concentration:

DNA yield depends on these major factors:

1. Volume of Elution Buffer
2. The way elution buffer is applied to the column
3. Incubation of elution buffer on column


Tips Improving Yield of Gel Extraction:

  • TAE buffer is preferred over TBE if the gel extract is used for downstream enzymatic application.
  • DNA recovery may be higher in TAE compared to TBE.
  • Small DNA fragments give higher recovery compared to larger fragments.
  • Higher recovery is observed when binding step is done at slow centrifugation speed.
  • Incubating the elution buffer in column for upto 5 minutes can increase the yield.

Sunday, June 23, 2019

Dot Blot Technique: Principle, Procedure & Advantages

What is Dot Blot?

Dot Blot is a cheaper, easier and faster technique to detect the presence of Proteins and Nucleic Acids in a biological sample. Due to the simplicity of the technique it widely used as a ideal diagnostic tool.

This post covers the Protein Dot Blot Technique.

Dot Blot Principle

Dot Blot works based on the immunodetection principle for identifying specific protein.


Protein Dot Blot
Dot Blot - Principle
Dot Blot - Technique: Overview

Proteins are immobilized on to a protein binding membrane, PVDF or Nitrocellulose. After Protein Immobilization membrane is blocked to avoid non-specific binding, using blocking buffer.

Immobilized protein is then incubated with a primary antibody (for 30mins to 1 hour). The antibody is specific to the protein and is visualized either with a specific tag or with a secondary antibody. 

The secondary antibody generally will carry a tag that allow the visualization of protein.

Blots can be visualized using methods such as Chemiluminescence or Fluorescence.

Common Tags used in Dot Blots are enzymes conjugated to the secondary antibody. Upon addition of substrate, enzyme acts on the substrate to develop colour. Commonly preferred enzymes are HRP(Horse Radish Peroxidase) and Alkaline Phosphatase (AP)

Advantages of Dot Blot over Western Blot


Non-Electrophoresis Technique:
Dot Blot is non-electrophoresis technique, means that one need not do protein electrophoresis. Proteins can directly immobilized on to the membrane.

Simple & Easy Method:
No messy work of PAGE run and gel to membrane transfers, proteins are directly immobilized on membrane, making this method much easier and faster.

Time:
It is a faster method, takes ~3hours, whereas western blot may even take up to 2 days. Including the protein electrophoresis and gel to membrane transfer optimization.

Dot Blot is not as informative as Western Blot, but it is inexpesive, quick and easy method for the simple protein detection. 

Sunday, June 16, 2019

Plastids - Introduction, Classification & Functions

Plastids - Introduction:

Plastids are membrane bound organelle found in all plant cells and some protists (euglina). Dr Ernst Haeckel discovered plastid.

Plastid Classification:

Plastids are classified into two types, they are:

1. Leucoplast
2. Chromoplast



Plastid Classification
Plastid Classification


Leucoplasts:

  • These are colourless plastids (non pigmented).
  • Leucoplasts do not have grana.


    • Leucoplasts are found in Roots, Rhizomes, Tubers, etc. ,parts of the plants which are not exposed to sunlight. 
    • Main function of Leucoplasts are storage of food.
    Based on their function Leucoplasts are further classified into 3 Types:

    a. Amyloplasts - Stores carbohydrates. Location: Potato Tuber, Wheat, Rice, etc

    b. Allueronplasts - They are also known as proteoplasts. They stores aluerone protein. Location: Maize, Pulses.

    c. Elaioplasts - Stores Fat. These can be found in castor seed, ground nut, etc.


    Chromoplasts:

    • These are coloured plastids (Colored due to presence of Pigments).
    • Chromoplasts have grana / Thylakoid
    • Chromoplasts are found in Leaf, Petals, Fruits, etc. , parts of the plants exposed to sunlight.
    • Main function of chromoplasts are photosynthesis, attraction of pollinating agents.
    Chromoplasts are further classified based on the type of pigment present:

    a. Chloroplasts - Green Pigmented plastid, found in Green leaves, green stem,etc

    b. Pheoplast - contains Xantophyll & Fucoxanthin. These are found in brown algae.
     - 
    c. Rhodoplast - contains red pigment called to phycoerythrin. these are found in red algae.

    Some of the other pigments present in plants are:

    1. Lycopin - Red pigment found in Tomato.
    2. Capsanthin - Red pigment found in Red Pepper.
    3. Carotenes - Orange / Red pigment found in carrots.

    Leucoplasts and Chromoplasts are interchangable, means that when lecuoplasts attains pigments it becomes chromoplasts and when the chromoplasts lose pigments it can become leucoplasts.