Southern, Northern, and Western blotting are techniques used to detect DNA, RNA, and proteins, respectively. Southern blotting involves transferring DNA fragments from a gel to a membrane where they can be detected using a probe. Northern blotting is used to identify RNA by separating RNA samples on a gel, blotting them to a membrane, and detecting them with a labeled probe. Western blotting identifies proteins by separating protein samples via electrophoresis, transferring them to a membrane, and detecting them using specific antibodies.
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
blotting techniques used to identify DNA, RNA and Protein and its Post-modification changes.
blotting techniques are of three basis types;
southern blotting
western blotting
eastern blotting
Probe labeling is defined as sequence use to search the mixture of nucleic acid for molecule containing complementary sequence.In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA
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 PPT discusses about the main types of Nucleic Acid Based Techniques - Blotting (Southern, Northen, Western)
Do Leave a comment if you liked the presentation, so that i can improve more and share more!
The methods used for DNA finger printing are the same Molecular markers...so for detailed note on the steps which is explained in DNA typing can be used to study the performance pf markers too...
blotting techniques used to identify DNA, RNA and Protein and its Post-modification changes.
blotting techniques are of three basis types;
southern blotting
western blotting
eastern blotting
Probe labeling is defined as sequence use to search the mixture of nucleic acid for molecule containing complementary sequence.In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA
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 PPT discusses about the main types of Nucleic Acid Based Techniques - Blotting (Southern, Northen, Western)
Do Leave a comment if you liked the presentation, so that i can improve more and share more!
The methods used for DNA finger printing are the same Molecular markers...so for detailed note on the steps which is explained in DNA typing can be used to study the performance pf markers too...
Blotting technique including Southern , Northern and Western blotting Rohit Mondal
he given ppt contains all the blotting techniques which is being studied by students in Biotechnology related subject and this PPT contais all blotting techniques in a very elaborative concise manner includes procedure principle application etc so which itwould help any bio student to take proper knowledge in this topic. I hope you will enjoy the content of the topic and would be able to grasp the topic properly
What is blotting? Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN.
Blotting
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier.
The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.
Types of blotting techniques
Southern Blotting
Northern Blotting
Western Blotting
A Southern blot is a method used
in molecular biology for detection of a specific DNA sequence in DNA samples.
Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
The method is named after its inventor, the British biologist Edwin Mellor Southern.
- Methods in Southern blotting
- Advantages and disadvantages
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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 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.
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5. INTRODUCTION
Blots are techniques for transferring of DNA, RNA &
proteins onto a carrier so they can be separated and
often follows the use of a gel electrophoresis.
6. TYPES
• Used to detect DNA
Southern
Blotting
• Used to detect RNA
Northern
Blotting
• Used to detect proteins
Western
Blotting
7. PRINCIPLE
Separation of protein or fragments of DNA or
RNA in given sample
Transfer to and immobilization on paper support
Binding of probe to target molecule on paper
Visualization of fragments bound with probe
10. Southern Blotting
Developed by Edward Southern in 1975
This technique requires:
DNA fragments by AGE
DNA fragments are blotted on to a strip of
nitrocellulose
Identification by probe
11. Definition
Southern blot is a method of transferring
DNA from an agarose gel to nitrocellulose
filter, on which the DNA can be detected
by suitable probe.
12. Procedure
DNA sample is digested by
restriction endonucleases.
Fragments of DNA are separated
by AGE or PAGE.
The restriction fragments present
in the gel are denatured by alkali .
13. Procedure
Then fragments are transferred to
nitrocellulose or nylon membrane
by blotting.
The filter is incubated under
hybridization conditions with a
specific radiolabelled probe.
Excess probe is washed away & the
probe bound to filter in detected
by autoradiography
17. Northern Blotting
This technique is used for the identification of RNA.
It was developed by James Alwine & George Stark at
stanford university 1979
18. Procedure
RNA is isolated
from several
biological
samples.
Samples are
loaded on gel &
RNA samples
are separated
according to
their size.
The gel is then
blotted on a
nylon
membrane or
nitrocellulose
by sandwich
arrangement.
19. Procedure
The membrane
is placed in a
dish with
hybridization
buffer
containing
labeled probe
The membrane
is washed to
remove
unbound
probe.
The labeled
probe is
detected by
autoradiograph
y
20. Applications
Study of gene expression at the mRNA level
Detection of mRNA transcript size
RNA
degradation
RNA splicing
Use to confirm
& check
transgenic
animals
RNA half life
21. Disadvantages
It is relatively less sensitive.
Detection with multiple probes is a
problem.
The quality & quantity of expression
is negatively affected by RNAses.
23. Procedure
A protein
sample is
subjected to
electrophoresis
on SDS.
Electro blotting
transfers the
protein from
gel to
nitrocellulose
membrane.
An antibody ,
specific for the
protein of
interest is
added to
membrane.
24. Procedure
After washing for removal of non specific AB1, second
antibody AB2 is added.
• AB2 recognizes the FC domain of primary antibody
and binds it.
AB2 is radioactively labeled,which allows to visualize
the protein AB1-AB2 complex.
27. Disadvantages
Requires specific antibody to a target protein, thus
many protein targets can’t be investigated due to lack
of specific antibodies.
• It might be more costly.
Very time consuming process.
• Can not analyze the sample of more than one gene.
