In a detail description of the two major blotting techniques. Right from its history to the result interpretation put forth in a concise way. Helps understand these protocols with ease.
In a detail description of the two major blotting techniques. Right from its history to the result interpretation put forth in a concise way. Helps understand these protocols with ease.
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!
In this presentation you will get a deep insight on the most important step of DNA fingerprinting that is the Quantitation of DNA.
You will understand what is DNA quantitation and also about the different techniques of DNA quantitation.
Nucleic acid techniques in diagnostic microbiologymohit kumar
in this presentation, you learn about the microbiological techniques which help in the molecular diagnosis of any single pathogens. with this, you aware of some commercially available kits for polymerase chain reaction both for realtime as well as conventional PCR and genome extraction kits.
This powerpoint explains about the nucleic acid hybridization, its principle, application and the assay methods. Also it gives clear picture about DNA probes, its sysnthesis, mechanism of probes and the detector system in DNA hybridization.
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
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...
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!
In this presentation you will get a deep insight on the most important step of DNA fingerprinting that is the Quantitation of DNA.
You will understand what is DNA quantitation and also about the different techniques of DNA quantitation.
Nucleic acid techniques in diagnostic microbiologymohit kumar
in this presentation, you learn about the microbiological techniques which help in the molecular diagnosis of any single pathogens. with this, you aware of some commercially available kits for polymerase chain reaction both for realtime as well as conventional PCR and genome extraction kits.
This powerpoint explains about the nucleic acid hybridization, its principle, application and the assay methods. Also it gives clear picture about DNA probes, its sysnthesis, mechanism of probes and the detector system in DNA hybridization.
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
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...
This report details the geological observations and interpretations made during a field investigation of the Kaptai Rangamati road-cut section, located in southeastern Bangladesh. The purpose of this report is to document the exposed rock units, their characteristics, and the geological structures present within the road cut.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
2. DNA QUANTIFICATION
Historical and commonly
used quantitation
methods include the
following:
•Yield gels
•Spectrophotometry
•Fluorometry
•Slot blot hybridization
•AluQuant®
•Quantitative PCR (qPCR)
3. Yield Gels
✓ semi-quantitative/qualitative assay
✓ The method consists of the electrophoresis of
DNA in an agarose gel matrix incorporating a
fluorescent intercalating dye such as ethidium
bromide (EtBr).
✓ The concentration of a sample can be
determined by comparing the intensity of the
fluorescence of the sample to that of the
calibration standards.
✓ Larger (undegraded) DNA fragments migrate
at a slower rate than those of lower molecular
weight.
✓ Degraded DNA will consist of lower molecular
weight fragments.
https://nij.ojp.gov/nij-hosted-online-training-courses/dna-extraction-and-
quantitation-forensic-analysts/quantitation/yield-gels
4.
5. Absorption Spectrophotometry
• The optical density (O.D.) of a solution
containing 50 µg/mL of double-stranded
DNA or 40 µg/µl of single-stranded
DNA is 1.00 at a wavelength of 260nm.
• The quality or purity of the sample can
be determined by comparing the
measurements at 260 and at 280 nm
(the wavelengths for which DNA and
protein absorb)
6.
7.
8. Fluorometry
• the molecules lose some of the absorbed
energy by irradiating light of a longer
wavelength. This property is known as
fluorescence.
• There are several dyes : fluorescence
enhancement when bound to double-
stranded DNA.
• PicoGreen® is one of the more common
fluorescent dyes that can be used in this
way.
• The fluorescence is easily measured using
a fluorometer.
11. Slot Blot Hybridization
• The majority of laboratories used the
commercially available QuantiBlot® kit,
which employs the following procedures:
Extracted DNA is
denatured to single-
stranded DNA is bound to
a positively charged nylon
membrane
a probe complementary to
the D17Z1 locus (present
in high quantities in higher
primates) is applied and
allowed to hybridize to
the DNA.
hybridized complex is
detection i.e
colorimetry/Chemiluminesc
ence,
12. Detection methods include:
•Colorimetry: tetramethylbenzidine
(TMB), which yields a blue color when
oxidized by hydrogen peroxide.
•Chemiluminescence: ACES (Gibco BRL)
and (Amersham Biosciences). The
chemiluminescent reactions cause the
release of photons that are captured on
film or a digital imaging device.
•Chemiluminescence is more sensitive
than colorimetry and can detect down to
10 to 20 pg of DNA
13.
14. AluQuant
• Alu sequences are abundant in the human genome numbering approximately
• Alu repeats (500,000 to 1,000,000 copies per genome. )were probed in the AluQuant
Human DNA Quantitation System.
denaturing the sample
and incubating it with the
AluQuant Enzyme and
AluQ Probe solutions
production of adenosine
triphosphate (ATP)
(correlated with the
amount of DNA present)
ATP was determined by
using a luminometer