this section helps students how to quanify the isolated DNA by spectrophotometer. specially life life science fields such as biotechnology, biology, and medical laboratory
Nucleic Acid Quantification Methods - DNA / RNA Quantificationajithnandanam
Nucleic acids are quantified to check the concentration and purity of DNA/RNA present in the solution mixture.it is important to know the concentration and purity of the nucleic acid for the use in further applications like PCR, restriction digestion etc. Spectrophotometric analysis is the most commonly used method of quantifying DNA, agarose gel electrophoresis can also be used to analyse the DNA sample for purity.
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
Nucleic Acid Quantification Methods - DNA / RNA Quantificationajithnandanam
Nucleic acids are quantified to check the concentration and purity of DNA/RNA present in the solution mixture.it is important to know the concentration and purity of the nucleic acid for the use in further applications like PCR, restriction digestion etc. Spectrophotometric analysis is the most commonly used method of quantifying DNA, agarose gel electrophoresis can also be used to analyse the DNA sample for purity.
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
This lectureis about DNA extraction from whole Blood presented by Tuba nafees she is msc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
lecture video is also there in youtube link:
https://www.youtube.com/watch?v=cGr__SuqYgY&t=409s
1. CENTRAL DOGMA OF MOLECULAR BIOLOGY
2. NUCLEIC ACID PREPARATION & APPLICATIONS
3. FUNDAMENTAL STEPS IN DNA PURIFICATION
4. ANALYSIS OF NUCLEIC ACIDS
5. STORAGE CONDITIONS
RNA, DNA Isolation and cDNA synthesis.pptxASJADRAZA10
Isolation, quantification of nucleic acids from wheat and synthesis of cDNA.
Introduction
List of Genotypes
DNA Isolation (CTAB method)
Qualitative check of DNA- Gel electrophoresis
Quantitative test of DNA- Spectrophotometer
Protocol for RNA Isolation
RNA Confirmation
Normalization of RNA
cDNA Synthesis
Protocol for DNA Isolation of plant
50-100mg (2-3) young leaves were collected, then washed with tap water followed by distilled water in petri dish.
Leaves were ground using ethanol sterilized mortar pestle for 15-20 sec, by taking 1mL extraction buffer.
1mL (1000μL) of extraction buffer was again added to collect paste from mortar pestle & then transferred to the 2 mL micro centrifuge tube.
The sample in the tube is incubated at 65°C in water bath for 35-45 mins. (Contents in the tube was mixed by inverting at an interval for 5-10 mins)
The tubes were cooled for 10 minutes in ice.
The sample of equal vol (2mL) was centrifuged @14,000 rpm for 10 mins.
After that the supernatant was transferred to new 2 mL centrifuge tube and equal volume (as of sample) of chloroform: Isoamyl alcohol (24:1) was added.
Then mixed gently for 5-7 mins by inverting the tubes.
Again centrifuged for 10 mins @10,000 rpm
After centrifugation, three layers were observed in the tube.
a) aqueous phase i.e. DNA+RNA
b) protein coagulate
c) organic phase i.e. Chloroform
Again the supernatant (aqueous phase) was collected in 1.5mL tube and equal volume of ice-cold isopropanol was added and stored in -20°C overnight.
Following day, tubes were again centrifuged @10,000rpm for 10 mins.
The supernatant was discarded without disturbing the DNA pellet.
70% ethanol is taken and 0.5mL of it was added to the sample and mixed by tapping for 5 mins.
Again centrifuged @10,000rpm for 10 mins and the supernatant was discarded.
Pellet (DNA Precipitate) was air dried for 10 mins.
Then dissolved in 50μL TE-1X Buffer and the sample was stored at -20°C.
1g of analytical grade Agarose was weighed.
100 mL of autoclaved 1X TBE was added in flask.
Now heated on the oven until the solution becomes transparent.
Solution was allowed to cool down to 60℃.
2 μL of Ethidium Bromide (EtBr) is added in the flask.
Melted agarose gel was poured into the casting tray along with comb.
Any bubble in the gel was removed.
After solidification of gel, comb was removed gently and then running buffer was added in the electrophoretic tank.
Once gel got solidified, it was transferred it into gel tank.
A parafilm was taken and on it 2μL loading dye and 3μL sample was taken, gently mixed with the pipette tip only.
Then the mixture (sample +loading dye) was loaded into the well.
Then electrophoretic unit was run at 90 volt for 50-55 mins.
After that gel was put into the Gel Doc to see the DNA band
(using UV light).
Bright colour band were observed as in the figure.
Few (100-150mg) young leaves were ground into fine powder using liquid Nitrogen.
This lectureis about DNA extraction from whole Blood presented by Tuba nafees she is msc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
lecture video is also there in youtube link:
https://www.youtube.com/watch?v=cGr__SuqYgY&t=409s
1. CENTRAL DOGMA OF MOLECULAR BIOLOGY
2. NUCLEIC ACID PREPARATION & APPLICATIONS
3. FUNDAMENTAL STEPS IN DNA PURIFICATION
4. ANALYSIS OF NUCLEIC ACIDS
5. STORAGE CONDITIONS
RNA, DNA Isolation and cDNA synthesis.pptxASJADRAZA10
Isolation, quantification of nucleic acids from wheat and synthesis of cDNA.
Introduction
List of Genotypes
DNA Isolation (CTAB method)
Qualitative check of DNA- Gel electrophoresis
Quantitative test of DNA- Spectrophotometer
Protocol for RNA Isolation
RNA Confirmation
Normalization of RNA
cDNA Synthesis
Protocol for DNA Isolation of plant
50-100mg (2-3) young leaves were collected, then washed with tap water followed by distilled water in petri dish.
Leaves were ground using ethanol sterilized mortar pestle for 15-20 sec, by taking 1mL extraction buffer.
1mL (1000μL) of extraction buffer was again added to collect paste from mortar pestle & then transferred to the 2 mL micro centrifuge tube.
The sample in the tube is incubated at 65°C in water bath for 35-45 mins. (Contents in the tube was mixed by inverting at an interval for 5-10 mins)
The tubes were cooled for 10 minutes in ice.
The sample of equal vol (2mL) was centrifuged @14,000 rpm for 10 mins.
After that the supernatant was transferred to new 2 mL centrifuge tube and equal volume (as of sample) of chloroform: Isoamyl alcohol (24:1) was added.
Then mixed gently for 5-7 mins by inverting the tubes.
Again centrifuged for 10 mins @10,000 rpm
After centrifugation, three layers were observed in the tube.
a) aqueous phase i.e. DNA+RNA
b) protein coagulate
c) organic phase i.e. Chloroform
Again the supernatant (aqueous phase) was collected in 1.5mL tube and equal volume of ice-cold isopropanol was added and stored in -20°C overnight.
Following day, tubes were again centrifuged @10,000rpm for 10 mins.
The supernatant was discarded without disturbing the DNA pellet.
70% ethanol is taken and 0.5mL of it was added to the sample and mixed by tapping for 5 mins.
Again centrifuged @10,000rpm for 10 mins and the supernatant was discarded.
Pellet (DNA Precipitate) was air dried for 10 mins.
Then dissolved in 50μL TE-1X Buffer and the sample was stored at -20°C.
1g of analytical grade Agarose was weighed.
100 mL of autoclaved 1X TBE was added in flask.
Now heated on the oven until the solution becomes transparent.
Solution was allowed to cool down to 60℃.
2 μL of Ethidium Bromide (EtBr) is added in the flask.
Melted agarose gel was poured into the casting tray along with comb.
Any bubble in the gel was removed.
After solidification of gel, comb was removed gently and then running buffer was added in the electrophoretic tank.
Once gel got solidified, it was transferred it into gel tank.
A parafilm was taken and on it 2μL loading dye and 3μL sample was taken, gently mixed with the pipette tip only.
Then the mixture (sample +loading dye) was loaded into the well.
Then electrophoretic unit was run at 90 volt for 50-55 mins.
After that gel was put into the Gel Doc to see the DNA band
(using UV light).
Bright colour band were observed as in the figure.
Few (100-150mg) young leaves were ground into fine powder using liquid Nitrogen.
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.
Lab 23 DNA Extraction and PurificationIsolation and purific.docxDIPESH30
Lab 2/3: DNA Extraction and Purification
Isolation and purification of nucleic acids is the most fundamental procedure in molecular biology. There are three basic steps involved:
1. Lyse (break open) the cells (and nuclei in eukaryotes) to release the DNA
2. Remove contaminants (proteins, lipids, carbohydrates, salts)
3. Preserve the integrity of the DNA (prevent degradation and shearing)
Step 1 can be accomplished in a number of ways, such as mechanical disruption (grinding, mincing), protein denaturation (detergents), and protein degradation (via proteases). These can be used singly or in combination depending on the type of biological sample you are starting with. Grinding the samples provides more surface area for the denaturants/proteases to interact with the cellular proteins, thus speeding up the denaturation process. We used liquid nitrogen (N2) and protein degradation (Proteinase K) in lab 2. Various salts are included in a cell lysis solution to stabilize the DNA by providing positive ions which insert between the negatively charged phosphates in the DNA backbone (creating a “salt bridge”). Buffers (such as Tris) also help to preserve DNA integrity by maintaining a neutral pH.
Once the cells have been lysed, contaminating proteins, lipids, etc. must be separated from the DNA. A widely used and efficient way to remove proteins from nucleic acids solutions is to extract with a 1:1 mixture of phenol and chloroform (CHCl3). Phenol and CHCl3 are both hydrophobic organic solvents that unfold proteins. When mixed with an aqueous DNA/protein solution and then centrifuged, the denatured proteins are selectively partitioned into the denser organic phase, while the DNA (plus RNA and salt) remains in the aqueous phase. This procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one. Additionally, chloroform removes any lingering traces of phenol from the nucleic acid preparation (which would interfere with later applications). Since the aqueous phase contains RNA and salt in addition to the DNA, phenol:CHCl3 extraction is followed by ethanol (EtOH) precipitation. DNA (a polar molecule) is soluble in water (also polar) because the water molecules intercalate into the phosphate backbone of the DNA and thus maintain it in a soluble state, but DNA is insoluble in 95% EtOH (nonpolar). Water molecules have a higher affinity for the EtOH than the DNA, so when you add EtOH and salt [10 M ammonium acetate (NH4Ac); pH 5.2], Na+ ions replace water in the DNA backbone, essentially removing the water molecules, and the DNA is forced out of solution (precipitates). After precipitating with 95% EtOH, the DNA is “washed” in 70% EtOH to remove the salt. Since 70% EtOH contains 30% water, the salt, having a greater affinity for the water than the DNA, remains in the EtOH, and the DNA is forced out.
The final step in the purification process is to preserve the DNA in a sta ...
You are asked to determine DNA content of a liver tissue. Describe h.pdfarwholesalelors
You are asked to determine DNA content of a liver tissue. Describe how you will porceed in
finding amount of DNA in the sample
Take a piece of live tissue and subject it to detergent solution i.e. SDS (sodiumdodecyl sulfate)
so that the cell membrane of the cells will be removed out through micelle formation & then
alcohol is added as DNA soluble in water. This is followed by the formation of aqueous phase
with DNA, precipitate layer with protein and bottom organic layer. Now the purity of DNA
sample is calculated to know the “total amount of DNA in the sample as ng DNA / µg dry
weight”. There are various processes applicable to determine total DNA content using either
cloning, qPCR, sequencing. However, initial purity of DNA can be calculated by measuring
absorbance or OD260/OD280 ratio at 280nm using “spectrophotomemeter” (UV-Visible). The
range of OD with absorption of UV light between 1.8 to 2.0 denotes the presence of DNA.
Lower than this OD ratio, denotes the presence of proteins. The another method of examining the
sample is through & this method is useful to deterimine total DNA concentration by use of the
fluorochrome Hoechst 33258 & filters, & scanning fluorescence spectrophotometer at emission
at 458 nm.
Solution
You are asked to determine DNA content of a liver tissue. Describe how you will porceed in
finding amount of DNA in the sample
Take a piece of live tissue and subject it to detergent solution i.e. SDS (sodiumdodecyl sulfate)
so that the cell membrane of the cells will be removed out through micelle formation & then
alcohol is added as DNA soluble in water. This is followed by the formation of aqueous phase
with DNA, precipitate layer with protein and bottom organic layer. Now the purity of DNA
sample is calculated to know the “total amount of DNA in the sample as ng DNA / µg dry
weight”. There are various processes applicable to determine total DNA content using either
cloning, qPCR, sequencing. However, initial purity of DNA can be calculated by measuring
absorbance or OD260/OD280 ratio at 280nm using “spectrophotomemeter” (UV-Visible). The
range of OD with absorption of UV light between 1.8 to 2.0 denotes the presence of DNA.
Lower than this OD ratio, denotes the presence of proteins. The another method of examining the
sample is through & this method is useful to deterimine total DNA concentration by use of the
fluorochrome Hoechst 33258 & filters, & scanning fluorescence spectrophotometer at emission
at 458 nm..
this section helps students how to isolate DNA from various sources. specially life life science fields such as biotechnology, biology, and medical laboratory
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Richard's aventures in two entangled wonderlandsRichard 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.
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.
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 .
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
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.
2. University of Gondar
Institute of Biotechnology
Techniques in Biotechnology (Biot.602)
Lecture 4
DNA quantification & purity determination
3. 3
Reliable measurement of DNA concentration is important
for many applications
DNA quantity and quality can be assessed using several
different methods include:
Absorbance by spectrophotometer or Nanophotometer.
Agarose gel electrophoresis .
Absorbance: is the most common easies to
determine DNA yield and purity.
Tadele T, April 2010 E.C
4. Quality of DNA using spectrophotometer
• An instrument employed to measure the amount of
light that a sample absorbs.
4Tadele T, April 2010 E.C
5. 5
The rings of the bases (A, C, G, T, U)
are made up of alternating single
and double bonds.
Such ring structures absorb in
the U.V.
Each of the four nucleotide
bases has a slightly different
absorption spectrum, and
The spectrum of DNA is
the average of them.
Tadele T, April 2010 E.C
6. ◦ DNA UV absorbance at 260nm.
◦ protein >> at 280nm.
◦ Carbohydrate >> at 230nm.
◦ Any insoluble light-scattering components……. absorbance at
320 nm.
Note: Nucleic acids absorb light at 260 nm ,the A260 reading should
be between 0.1–1.0. The spectrophotometer is most accurate when
measurements are in the range of 0.1–1.0.
However, DNA is not the only molecule that can absorb UV-
light at 260nm.
Since RNA also has a great absorbance at 260nm will
contribute to the total measurement at 260nm
6Tadele T, April 2010 E.C
7. The ratio of the absorbance at 260 nm/280 nm is a
measure of the purity of a DNA; it should be between
1.7 and 2.0.
If < 1.7, the nucleic acid preparation may be contaminated with
protein. Use protinase K to remove protein.
If > 2.0 indicates RNA contamination. RNase should be used to
remove the contaminating RNA.
DNA Purity (A260/A280) = (A260 reading – A320 reading)
/(A280 reading – A320 reading)
7Tadele T, April 2010 E.C
8. The ratio of the absorbance at 260 nm/320 nm is a measure
of the purity of a DNA sample from organics and/or salts;
it should be about 2.0.
Low A260/A320 ratio indicates contamination by organics
and/or salts.
The absorbance reading indicates how much the sample is pure.
8Tadele T, April 2010 E.C
9. Quantification of DNA by spectrophotometry.
Using TE buffer as the diluent,
Make an appropriate dilution of your DNA depending on
the size of the cuvettes available (e.g. for 1ml cuvettes,
dilute 10 microliter DNA solution in 990 micro liters of
TE).
Determine the absorbance of DNA at 260 nm using TE as the
reference solution (i.e. as a blank).
9Tadele T, April 2010 E.C
10. Using a conversion factor :
◦ one OD at 260 nm is equivalent to
Multiply the absorbance reading by
the conversion factor and
the dilution factor to find the concentration of nucleic
acid.
Pure DNA Concentration (microg/ml) =
(A260 reading – A320 reading) x dilution factor x 50microg/ml
10Tadele T, April 2010 E.C
11. Total yield is obtained by multiplying the
DNA concentration by the final total purified
sample volume.
DNA Yield (microgram/ml) = DNA
Concentration x Total Sample Volume
11Tadele T, April 2010 E.C
12. Problem. From a small culture, you have purified the DNA of a
recombinant plasmid. Then you have resuspended the DNA in a
volume of 50 µL TE. You dilute 20 µL of the purified DNA sample
into a total volume of 1000 µL distilled water. You measure the
absorbance of this diluted sample at 260 nm and 280 nm and obtain
the following readings.
A260 --- 0 . 5 5 0
A280 - 0 . 3 2 4
a) What is the DNA concentration of the 50 µL plasmid prep?
b) How much total DNA was purified by the plasmid prep
procedure?
c) What is the A260/280 ratio of the purified DNA?
12Tadele T, April 2010 E.C
13. 13
Don’t need dilution
The volume required for measurement 3-5
microliters
The concentration given in nanogram
microliters.
Tadele T, April 2010 E.C
14. 14
Quality of DNA extracted is assessed using
the following simple protocol:
Mix 3µL of DNA with 12µL of loading
Dye
Load this mixture into a 1% agarose gel
Stain with ethidium bromide
Electrophorese at 70–80 volts, 45–90
minutes.
Tadele T, April 2010 E.C
15. Checking for Degradation DNA
Running your sample through an agarose gel is a
common method for examining the extent of DNA
degradation.
Smearing indicates
DNA degradation or
Too much DNA loaded.
15Tadele T, April 2010 E.C
16. 16Tadele T, April 2010 E.C
Good quality DNA should
migrate as a high molecular
weight band, with little or no
evidence of smearing.