Southern blotting is a technique used to detect specific DNA sequences. It involves separating DNA fragments by size via gel electrophoresis, transferring them to a membrane, and using a labeled probe to hybridize to the target sequence. The probe binds only to complementary DNA fragments, allowing their detection and location on the membrane via autoradiography. Key steps include restriction enzyme digestion of DNA, gel electrophoresis, membrane transfer, probe hybridization, and x-ray film development to visualize hybridized fragments. Southern blotting is used for applications like mutation detection, gene rearrangement studies, and forensic analysis.
This PPT discusses about the main types of Nucleic Acid Based Techniques - Blotting (Southern, Northen, Western)
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Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
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!
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
WHAT IS BLOTTING?
Blotting is a technique for detecting any macromolecules that we deal with like DNA, RNA or proteins, which are initially present in a complex mixture.
TYPES OF BLOTTING:
Southern Blotting
Northern Blotting
Western Blotting
NORTHERN BLOTTING
A northern blotting is a laboratory method used to detect specific RNA molecules among a mixture of RNA (mRNA).
The technique was developed in 1979 by James Alwine and his colleagues.
Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell type in order to measure the expression of particular genes.
Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence.
The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However the entire process is commonly referred to as northern blotting.
PROCEDURE
1.RNA isolation:
2.Separation of RNA using gel electrophoresis:
3.BLOTTING:
4.Hybridization with labelled probe:
5.WASHING OFF EXCESS PROBES
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.
WHAT IS BLOTTING?
Blotting is a technique for detecting any macromolecules that we deal with like DNA, RNA or proteins, which are initially present in a complex mixture.
TYPES OF BLOTTING:
Southern Blotting
Northern Blotting
Western Blotting
NORTHERN BLOTTING
A northern blotting is a laboratory method used to detect specific RNA molecules among a mixture of RNA (mRNA).
The technique was developed in 1979 by James Alwine and his colleagues.
Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell type in order to measure the expression of particular genes.
Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence.
The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However the entire process is commonly referred to as northern blotting.
PROCEDURE
1.RNA isolation:
2.Separation of RNA using gel electrophoresis:
3.BLOTTING:
4.Hybridization with labelled probe:
5.WASHING OFF EXCESS PROBES
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.
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
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
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
How to Split Bills in the Odoo 17 POS ModuleCeline George
Bills have a main role in point of sale procedure. It will help to track sales, handling payments and giving receipts to customers. Bill splitting also has an important role in POS. For example, If some friends come together for dinner and if they want to divide the bill then it is possible by POS bill splitting. This slide will show how to split bills in odoo 17 POS.
The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
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”.
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?
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
2. BLOTTING
is a method of transferring proteins, DNA or RNA onto a carrier (for
example, a nitrocellulose or nylon membrane.
Types
1. Southern Blotting (DNA)
2. Northern Blotting (RNA)
3. Western Blotting (Proteins)
4. Eastern Blotting (Post translational modifications of proteins)
3. SOUTHERN BLOTTING
Discovered by Edwin Southern , 1975
Purpose:
1. Used for detection of a specific DNA sequence in DNA samples.
2. Southern blotting technique is used to isolate a particular DNA
3. To study mutation and gene rearrangement.
4. This technique is used in phylogenetic studies, paternity & maternity
analysis and forensic studies.
4. Principle:
KEY FEATURE: Hybridization
Hybridization: It is the process of forming a double stranded
DNA molecule between a single-stranded DNA probe and a single-stranded
target DNA.
Separation of DNA fragments by gel electrophoresis
followed by the identification by labeled probe hybridization.
The DNA fragments are separated based on their size and charge during
electrophoresis
5. 1.Extract and Purify Dna
2.Digest the DNA by restriction enzyme-
fragments formed
3.Separation of fragments by Gel
Electrophorsis
4.Denature the DNA by Alkali treatment -
single stranded Dna
5.Transfer the denatured DNA to the
membrane..
6. 6.Add labeled probe for hybridization to
take
7. Wash off unbound probe;
8. Autoradiograph
7. STEPS
1.Extract and purify DNA from cells
• Isolate the DNA from the rest of the cellular material in the
nucleus.
• Isolated DNA is purified from solution by alcohol precipitation.
2. DNA is restricted with enzymes.
9. 4. Denature DNA
DNA bands in the gel are denatured into single stranded form by
alkali treatment (0.5 M NaOH).
5.Transfer to nitrocellulose paper:
Place the gel on top of a buffer saturated filter paper
Lay the Nitrocellulose filter membrane on the top of the gel
Place some dry filter paper on top of this membrane. From the bottom
filter paper , buffer move by capillary action through the gel , carrying
with it the denatured DNA present in the gel .
DNA get trapped in the nitrocellulose membrane as the buffer passes
through it because of its greater affinity → replica pattern.
10. Bake nitrocellulose membrane with ssDNA bands at 80 ⁰ C for 2-3 hours-
to fix the DNA permanently on the membrane.
Nitrocellulose membrane with the replica of DNA bands from the Agarose
gel is used for hybridization with labelled DNA probe –
11. 6. Add labeled probe for hybridization:
Nitrocellulose sheet is moistened with a minimal quantity of solution
containing a ³²P labelled ssDNA probe that is complementary in sequence
to the DNA of interest
The probe hybridizes with the complementary Dna.
A hybridization probe is a fragment of DNA or RNA of variable length
(usually 100–10000 bases long) which can be radioactively or fluorescently
labeled.
It is used in DNA or RNA samples to detect the presence of nucleotide
substances that are complementary to the sequence in the probe
12. 7. Wash the unbound probe.
8. Autoradiograph:
Place it over a sheet of X –ray film – the positions of the molecules that
are complementary to the radioactive probe are indicated by a
darkening of the developed film.
