This document discusses different methods for nucleic acid purification. It describes the purpose of nucleic acid purification as extracting ample amounts of DNA or RNA from a limited source while reducing contaminants. Three major purification methods are discussed: spin column-based purification using silica membranes, the Boom method which uses silica beads, and phenol-chloroform extraction which separates nucleic acids from proteins into aqueous phases. Recent trends include automation, commercial kits, and use of chaotropic agents to improve nucleic acid yields and purity.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
b pharma 6th sem
nucleic acid extraction and quantification
pharmaceutical biotechnology
Introduction
Purpose
Isolation
Methods of isolation
Basic steps for DNA extraction
Organic extraction
Inorganic extraction
salting out
There are 'n' number of DNA isolation methods depending on the sample type, final use of DNA product, etc. This presentation gives an overall idea about different methods of DNA isolation in a simplified way.
Techniques of DNA Extraction, Purification and QuantificationBHUMI GAMETI
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
2011 course on Molecular Diagnostic Automation - Part 1 - DNA ExtractionPatrick Merel
2011 course on Molecular Diagnostic Automation - Part 1 - Nucleic Acid Extraction.
This is from early 2011. Prices and Specifications of instruments may have changed.
Part 1 of 3
Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptxEduSkills OECD
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The Art Pastor's Guide to Sabbath | Steve ThomasonSteve Thomason
What is the purpose of the Sabbath Law in the Torah. It is interesting to compare how the context of the law shifts from Exodus to Deuteronomy. Who gets to rest, and why?
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
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Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
The Indian economy is classified into different sectors to simplify the analysis and understanding of economic activities. For Class 10, it's essential to grasp the sectors of the Indian economy, understand their characteristics, and recognize their importance. This guide will provide detailed notes on the Sectors of the Indian Economy Class 10, using specific long-tail keywords to enhance comprehension.
For more information, visit-www.vavaclasses.com
How to Make a Field invisible in Odoo 17Celine George
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Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
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We all have good and bad thoughts from time to time and situation to situation. We are bombarded daily with spiraling thoughts(both negative and positive) creating all-consuming feel , making us difficult to manage with associated suffering. Good thoughts are like our Mob Signal (Positive thought) amidst noise(negative thought) in the atmosphere. Negative thoughts like noise outweigh positive thoughts. These thoughts often create unwanted confusion, trouble, stress and frustration in our mind as well as chaos in our physical world. Negative thoughts are also known as “distorted thinking”.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
Ethnobotany and Ethnopharmacology:
Ethnobotany in herbal drug evaluation,
Impact of Ethnobotany in traditional medicine,
New development in herbals,
Bio-prospecting tools for drug discovery,
Role of Ethnopharmacology in drug evaluation,
Reverse Pharmacology.
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The map views are useful for providing a geographical representation of data. They allow users to visualize and analyze the data in a more intuitive manner.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
1. Topic : Nucleic Acid Purification
Presented by: D.Sairam
Course: Bioanalytical and
Instrumentation-I
Course Code: BSBT-202
Course Instructor: Dr.Subhabrata Kar
2. Overview of the Presentation
• Introduction ( Nucleic Acids)
• Purpose of Purification
• Methods of Nucleic Acid Purification
• Research Trends in Nucleic Acid
Purification
• References
3. Introduction
Nucleic acids are polymolecules, or large biomolecules, essential for all
known forms of life. Nucleic acids, which include DNA
(Deoxyribonucleic acid) and RNA (Ribonucleic acid), are made from
monomers known as nucleotides.
DNA purification is a technique that removes impurities and unused reagents
from samples after enzymatic reactions, such as PCR.
There are three major large scale processes of DNA Extraction namely:
• Spin Column-based Nucleic acid Purification
• Boom method (Boom nucleic acid extraction method)
• Phenol–chloroform extraction
4. Purpose of Nucleic Acid Purification
• It usually determines the success or failure of all your
immediate and downstream experimentation.
• Extract ample amounts of your genomic and/or plasmid DNA
sample from a limited source to satisfy the requirements of your
research.
• Purify it to reduce the amount of contaminants that can
compromise the results of your research and shorten the shelf-
life of your precious sample.
5. Boom Method
(Boom Nucleic Acid Extraction Method)
This method is one of the most widespread method for isolating nucleic acids
from biological samples and is known as a simple, rapid, and reliable method
for the small-scale purification of NA from Biological sample.
The crux of the method of this method is the use of Silica beads, capable of
binding the NA in the presence of a chaotropic substance.
This method is said to have been developed and invented by Willem R. Boom
in 1990.
Amorphous silicon oxide and glass powder, Alkyl silica, Aluminium silicate
(zeolite), or, activated silica with -NH2 (Amino) are suitable as nucleic acid
binding solid phase material.
Today, the embodiments of Boom method, characterized by "utilizing the
magnetic beads (silica beads are magnetic beads)" is widely used.
6.
7. Spin Column-based Nucleic acid Purification
Genesis of the Process: DNA binds to silica, glass particles or to unicellular algae
called diatoms which shield their cell walls with silica. This property was used to
purify nucleic acid using glass powder or silica beads under alkaline conditions.
It is a solid phase extraction method to quickly purify nucleic acids. This method
relies on the fact that nucleic acid will bind to the solid phase of silica under
certain conditions.
The stages of the method are:
• Lyse – The cells of a sample are broken open with a lysis procedure.
• Bind – A buffer solution is then added to the sample along with ethanol or
isopropanol. This forms the binding solution. The binding solution is transferred to
a spin column and the column is put in a centrifuge. The centrifuge forces the
binding solution through a silica gel membrane that is inside the spin column. If
the pH and salt concentration of the binding solution are optimal, the nucleic acid
will bind to the membrane as the solution passes through.
8. • Wash – The flow-through is removed and a wash buffer is added to the
column. The column is put in a centrifuge again, forcing the buffer through
the membrane. This washes any remaining impurities from the membrane,
leaving only the nucleic acid bound to the silica gel.
• Elute – The wash buffer is removed and an elution buffer (or simply water) is
added to the column. The column is put in a centrifuge again, forcing the
buffer through the membrane. The elution buffer removes the nucleic acid
from the membrane and it is collected from the bottom of the column.
9.
10. Phenol–Chloroform extraction
This is a liquid-liquid extraction technique in biochemistry and molecular biology
for purifying nucleic acids and eliminating proteins.
In brief, aqueous samples are mixed with equal volumes of a phenol: chloroform
mixture.
After mixing, the mixture is centrifuged and two distinct phases are formed,
because the Phenol: Chloroform mixture is immiscible with water.
The aqueous phase is on top because it is less dense than the organic phase
(Phenol: Chloroform). The proteins will part to the lower organic phase while the
nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in
the upper aqueous phase.
The upper aqueous phase is pipetted off and care is taken to avoid pipetting any of
the organic phase or material at the interface. This procedure is often performed
multiple times to increase the purity of the DNA
11.
12. Recent Trends in DNA Purification
Refinements of the technique include adding a chelating agent to sequester
divalent cations, such as Mg2+ and Ca2+, which prevents enzymes like Dnase
from degrading the DNA.
With Advancements in Robotics have resulted in scientists contemplating the
use of Robots for complex processes such as DNA Purification.
We have also seen certain companies such as QIAGEN, Promega, Master
Pure etc. manufacture kits for DNA Purification.
Compounds such as Guanidinium Thiocyanate or Guanidinium
Hydrochloride as the chaotropic agent.