This document describes the process of preparing and isolating genomic DNA from bacterial cells. It involves 4 main steps:
1) Growing and harvesting bacterial cells in nutrient broth media. Common media used are M9 and Luria-Bertani broth.
2) Preparing a cell extract by lysing the bacterial cells using enzymes like lysozyme and detergents like SDS.
3) Purifying the DNA from other cell components like proteins and RNA. This is done using phenol-chloroform extraction and protease/RNase digestion. Ion-exchange chromatography can also be used.
4) Concentrating the purified DNA using ethanol precipitation, which causes the long DNA strands to precipitate out of
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.
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.
pBluescript is an example of a combination between plasmids and phages (phagemids).
Phagemids represent a hybrid type of class of vectors that serve to produce single-stranded DNA.
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.
in this presentation, what are the steps and strategies involved the gene cloning and i was focused only on the 1st two steps of gene cloning.they are generation of foreign DNA molecules and selection of suitable vectors.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
It is common for students to use kits without knowing exactly what the different solutions/buffers are doing or what are they composed of. This automate attitude is wrong, thus, a proper discussion over the ins and outs of DNA extraction kits is imperative. The Toxicologist Today gives a little help, if you know more help us by commenting.
pBluescript is an example of a combination between plasmids and phages (phagemids).
Phagemids represent a hybrid type of class of vectors that serve to produce single-stranded DNA.
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.
in this presentation, what are the steps and strategies involved the gene cloning and i was focused only on the 1st two steps of gene cloning.they are generation of foreign DNA molecules and selection of suitable vectors.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
It is common for students to use kits without knowing exactly what the different solutions/buffers are doing or what are they composed of. This automate attitude is wrong, thus, a proper discussion over the ins and outs of DNA extraction kits is imperative. The Toxicologist Today gives a little help, if you know more help us by commenting.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
molecular biology techniques -jaypee university of information technology- ra...RAVI RANJAN
molcular biology techniques- ravi ranjan lb-
contents- basic molecular biology techniques - DNA and RNA isolation from plant sample, nanodrop technique, pcr and cloning.
this section helps students how to isolate DNA from various sources. specially life life science fields such as biotechnology, biology, and medical laboratory
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
How to Create Map Views in the Odoo 17 ERPCeline George
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.
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”.
This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
He discussed the concept of quality improvement, emphasizing its applicability to various aspects of life, including personal, project, and program improvements. He defined quality as doing the right thing at the right time in the right way to achieve the best possible results and discussed the concept of the "gap" between what we know and what we do, and how this gap represents the areas we need to improve. He explained the scientific approach to quality improvement, which involves systematic performance analysis, testing and learning, and implementing change ideas. He also highlighted the importance of client focus and a team approach to quality improvement.
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?
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.
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
The French Revolution Class 9 Study Material pdf free download
Preparation and isolation of genomic
1. Molecular
Genetics
M.Mubashar ali
Roll# 01
BS zoology
6th Semester
+923026243208
Preparation
and isolation
of genomic
DNA
1
2. CONTENTS
Introduction
Apparatus
Chemical
Procedure (Steps)
1. Growing and harvesting a bacterial culture
2. Preparation of a cell extract
3. Purification of DNA from a cell extract
4. Concentration of DNA samples
2
3. INTRODUCTION
Bacteria are prokaryotic organisms having DNA
without any membrane bounded nucleus ,their DNA
is circular and present in cytoplasm .This DNA is
called Genomic DNA
Genomic DNA will often be required as a source of
material from which to obtain genes to be cloned.
Genomic DNA may be obtain from a culture of
bacteria , from a plant, from animal cells.
3
4. Apparatus
Test tubes
Petri dish
Wire loop
Autoclave
Beaker
Conical flask
Physical balance
Stirring rod
Centrifuge
4
5. CHEMICALS
For culturing of bacteria:
Nutrient agar ,Nutrient broth( M9 medium and Luria-
Bertani (LB) medium.
For isolation:
By lysozyme, 50ml ethylenediamine tetra acetate
(EDTA), or a combination of both.
Sodium dodecyl sulphate (SDS)
1 : 1 phenol( pH 8.0) and chloroform
Proteinase K enzyme
Ribonuclease enzyme
Chromatographic matrix or resin
Monovalent cations such as sodium ions
Ethanol
5
6. PROCEDURE
The procedure for genomic DNA preparation from a
culture of bacterial cells can be divided into four
stages(Figure 1):
1. Growing and harvesting a bacterial culture
2. Preparation of a cell extract
3. Purification of DNA from a cell extract
4. Concentration of DNA samples
6
7. Fig 1: The basic steps in preparation of total cell DNA
from a culture of bacteria.
7
8. STEPS
1.Growing and harvesting a bacterial culture
Culture medium must provide a balanced mixture of the
essential nutrients at concentrations that will allow the
bacteria to grow and divide efficiently
Two type of liquid mediums used for growing
M9 medium
defined mediumin which all the components are known.
It provide essential elements such as nitrogen, magnesium,
and calcium, as well as glucose to supply carbon and
energy.
Have growth factors such as trace elements and vitamins.
The bacterial culture has to be grown under precisely
controlled conditions.
8
9. Luria-Bertani (LB) medium
Undefined medium, meaning that the precise
identity and quantity of its components are not
known.
Contain tryptone and yeast extract, are complicated
mixtures of unknown chemical compounds.
Tryptone supplies amino acids and small peptides.
Yeast extract (a dried preparation of partially digested
yeast cells) provides the nitrogen requirements, along
with sugars and inorganic and organic nutrients.
LB need no further supplementation and support the
growth of a wide range of bacterial species.
9
10. The composition of two typical media for the growth of
bacterial cultures.
10
11. Harvesting a bacterial culture
Performed by spinning the culture in a centrifuge
Fairly low centrifugation speeds will pellet the bacteria
at the bottom
Bacteria from a 1000 ml re suspended into a volume of
10 ml or less.
11
12. 2.Preparation of a cell extract
The bacterial cell is enclosed in a cytoplasmic
membrane and surrounded by a rigid cell wall
cell lysis is brought about by exposure to chemical
agents
involves one agent attacking the cell wall and another
disrupting the cell membrane
brought about by lysozyme, ethylenediamine
tetraacetate (EDTA), or a combination of both.
Lysozyme is present in egg white and in secretions
such as tears and saliva, and which digests the
polymeric compounds that give the cell wall its
rigidity.
12
13. EDTA (Ethylene Diamine tetra accetic acid)a chelating
agent removes magnesium ions that are essential for
preserving the overall structure of the cell envelope,
and also inhibits cellular enzymes that could degrade
DNA.
Detergent such as sodium dodecyl sulphate (SDS) is
also added it aid the process of lysis by removing lipid
molecules and thereby cause disruption of the cell
membranes
13
14. the final step in preparation of a cell extract is removal
of insoluble cell debris
cell extract can be pelleted by centrifugation leaving
the cell extract as a reasonably clear supernatant.
14
15. 3.Purification of DNA from a cell
extract
In addition to DNA, a bacterial cell extract contains
significant quantities of protein and RNA and should
be removed
Two methods are used for removing protein and RNA
Removing contaminants by organic
extraction and enzyme digestion
to deproteinize a cell extract is to add phenol or a 1 : 1
mixture of phenol and chloroform
15
16. The layers then separated by centrifugation,
precipitated protein molecules are left as a white
coagulated mass at the interface between the
aqueous and organic layers
The aqueous solution of nucleic acids can then be
removed with a pipette.
Several phenol extractions one after the other
because protein content is so great that a single phenol
extraction is not sufficient to completely purify the
nucleic acids
16
17. It results in a certain amount of breakage of the DNA
molecules
the cell extract treated with a protease such as
pronase or proteinase K before phenol extraction.
This enzymes break polypeptides down into smaller
units, which are more easily removed by phenol
Effective way to remove the RNA is with the enzyme
ribonuclease, which rapidly degrades these
molecules into ribonucleotide subunits.
17
19. Using ion-exchange chromatography to purify
DNA from a cell extract
Separates molecules according to how tightly they
bind to electrically charged particles present in a
chromatographic matrix or resin
DNA and RNA are both negatively charged, as are
some proteins, and so bind to a positively charged
resin
The electrical attachment is disrupted by salt, removal
of the more tightly bound molecules requiring higher
concentrations of salt
The resin in a glass or plastic column and then add the
cell extract to the top
19
20. The extract passes through the column, and because
this extract contains very little salt all the negatively
charged molecules bind to the resin
If a salt solution of gradually increasing concentration
is now passed through the column, the different types
of molecule will become unbound in the sequence
protein, RNA, and finally DNA.
Different salt solutions are used to unbound the
protein and RNA, leaving just the DNA bound,
followed by a second of a higher concentration which
unbound the DNA, now free from protein and RNA
contaminants.
20
23. 4.Concentration of DNA samples
It is important to increasing the DNA concentration
frequently used method of concentration is ethanol
precipitation
In the presence of salt of monovalent cations such as
sodium ions and at a temperature of −20°C or less,
absolute ethanol efficiently precipitates polymeric nucleic
acids
Ethanol precipitation has the added advantage of leaving
short-chain and monomeric nucleic acid components in
solution
A glass rod pushed through the ethanol into the DNA
solution. When the rod is removed,DNA molecules adhere
and can be pulled out of the solution in the form of a long
fiber
The precipitate can be collected by centrifugation
23
24. Collecting DNA by ethanol precipitation. (a) Absolute ethanol is layered on
top of a concentrated solution of DNA. Fibers of DNA can be withdrawn with a
glass rod. (b) For less concentrated solutions ethanol is added (at a ratio of 2.5
volumes of absolute ethanol to 1 volume of DNA solution) and precipitated
DNA collected by centrifugation.
24