This document provides instructions for extracting genomic DNA from whole blood using a solution-based extraction kit. The summary is as follows:
The procedure involves lysing red blood cells and white blood cells to isolate genomic DNA from whole blood. The cells are lysed using specific lysis buffers, and proteins and other contaminants are removed through precipitation and washing steps. High molecular weight genomic DNA is then purified through isopropanol precipitation. The extracted DNA can be analyzed through agarose gel electrophoresis and spectrophotometry to assess quality and concentration. High quality genomic DNA will appear as an intact band on the gel without RNA contamination and have an A260/280 ratio around 1.8, indicating pure DNA.
As you know, Sample preparation is a relevant step to get the success in your research. For this reason, Canvax™ Extraction kits are designed for a reliable, easy and fast purification of high-quality and high-purity genomic DNA/RNA from a wide range of starting materials, like Blood, Buccal Swab, Buccal Saliva, Tissues, Cultured cells, Stool samples, Plant tissues, Soil samples, Bacteria, Yeast, Plasmid, PCR mixtures, Agarose gel slices or Serum.
Our breakthrough and proprietary technologies HigherPurity™, CleanEasy™ and WideUSE™ improves the most common Extraction methods conferring the obtained DNA and RNA a proven optimal performance for all downstream applications, such as PCR, qPCR, NGS, Cloning, STR Analysis or Gene Expression.
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
As you know, Sample preparation is a relevant step to get the success in your research. For this reason, Canvax™ Extraction kits are designed for a reliable, easy and fast purification of high-quality and high-purity genomic DNA/RNA from a wide range of starting materials, like Blood, Buccal Swab, Buccal Saliva, Tissues, Cultured cells, Stool samples, Plant tissues, Soil samples, Bacteria, Yeast, Plasmid, PCR mixtures, Agarose gel slices or Serum.
Our breakthrough and proprietary technologies HigherPurity™, CleanEasy™ and WideUSE™ improves the most common Extraction methods conferring the obtained DNA and RNA a proven optimal performance for all downstream applications, such as PCR, qPCR, NGS, Cloning, STR Analysis or Gene Expression.
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
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
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.
The technique of molecular biology like DNA isolation, RNA isolation, PCR, Western blot, RFLP, etc was developed with development in science. This presentation includes the method of DNA and RNA isolation and their Quantification techniques.
DNA extraction is an important step in molecular assays and plays a vital role in obtaining highresolution results in gel-based systems, particularly in the case of cereals with high content of interfering components in the early steps of DNA extraction.This is a rapid miniprep DNA extraction method, optimized for rice, which was achieved via creating some modifications in present DNA extraction methods, especially in first step of breaking down and lyses of cell wall, and the use of cheap and frequent chemicals, found in every lab, in the next steps. The normal quality and quantity was obtained by the method. The PCR based assays also revealed the efficiency of the method.
The advantages of this method are: 1- it is applicable with both dry and fresh samples, 2- no need to large weight samples, 3- no need to liquid nitrogen and 4- easy, rapid and applicable in every laboratory.
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.
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
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.
The technique of molecular biology like DNA isolation, RNA isolation, PCR, Western blot, RFLP, etc was developed with development in science. This presentation includes the method of DNA and RNA isolation and their Quantification techniques.
DNA extraction is an important step in molecular assays and plays a vital role in obtaining highresolution results in gel-based systems, particularly in the case of cereals with high content of interfering components in the early steps of DNA extraction.This is a rapid miniprep DNA extraction method, optimized for rice, which was achieved via creating some modifications in present DNA extraction methods, especially in first step of breaking down and lyses of cell wall, and the use of cheap and frequent chemicals, found in every lab, in the next steps. The normal quality and quantity was obtained by the method. The PCR based assays also revealed the efficiency of the method.
The advantages of this method are: 1- it is applicable with both dry and fresh samples, 2- no need to large weight samples, 3- no need to liquid nitrogen and 4- easy, rapid and applicable in every laboratory.
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.
The extraction of DNA involves three main steps that are cell lysis, protein separation, and DNA purification. Cell lysis is usually performed by incubation of cell in buffer containing detergent and protease. Cellular proteins are salted out or phase separated using organic solvents. Finally DNA is isolated and purified either by alcohol precipitation or adsorption with silica and elution.
Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis .docxmoirarandell
Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis
2
Objective
· Digest DNA of pGLO plasmid using restriction endonuclease enzymes.
· Run an agarose gel to separate the DNA fragments.
Background
Restriction enzymes cut DNA at specific sites generating a number of different sized fragments. The size of the fragments will depend on the number of sites the plasmid has and the specific enzyme used. The number of fragments can be predicted by viewing the map of the plasmid
Gel electrophoresis is a means of separating DNA in an electrical field. DNA is negatively charged and so will move to the anode (+). Larger fragments will move slower through the agarose matrix than the smaller molecules. Agarose is a polysaccharide polymer derived from seaweed: it is a purified from agar by removing the agaropectin component. Fragments are visualized using ethidium bromide, which will glow orange when exposed to UV light.
Materials
Restriction digest
· Restriction enzymes: Nhe1 and EcoR1 (New England Biolabs) – (KEEP ON ICE)
· Plasmid prepared in lab 7
· NanoDrop Lite spectrophotometer
· Microfuge tubes – Sterile
· 37 C degree bath – block heater
· Sterile 10ul and 200ul tips
· Bleach bottles for cleaning bench
· 10X NE Cut Smart Buffer – comes with enzyme
· Nitrile gloves
· Sterile DI water
· Shaved ice
· Ice block for enzymes
Gel Electrophoresis
· Agarose
· Sterile miliQ Water
· 15 well comb
· 50x TAE buffer
· DNA ladder – diluted in sample buffer (1 KB)
· Gel loading dye
· Gel electrophoresis chamber
· Power supply
· Ethidium bromide
· Gel Sys – visualization system
_______________________________________________
Procedure
Restriction Digest of plasmid DNA
· Safety: Wear nitrile gloves – prevent DNAase from your hands affecting the reaction and protect yourself from ethidium bromide
· Clean the bench with bleach - prevents exogenous enzymes interfering you’re your digests.
· Use the NanoDrop to determine the amount of DNA in your plasmid prep. Use this information to calculate how much sample you need to pipette into the reaction mix.
· Label an Eppendorf tube ‘+’ and another ‘-‘
· Make up a reaction mix in both tubes as follows for one of your plasmid samples
· add 1ug of DNA from your plasmid prep
· 5ul of 10X NE Cut Smart Buffer
· Sterile DI water to make the reaction mix to 50ul
For the + tube
· DNA
x ul
· 10X NE Cut Smart Buffer
5ul
· Nhe1 (add last to + tube)
1ul
· EcoR1 (add last to + tube)
1ul
· Sterile DI water
To make final volume to 50ul
· Add the restriction enzymes last to the + tube ONLY
· Repeat with the other two plasmid samples
For the – tube
· DNA
x ul
· 10X NE Buffer
5ul
· Nhe1
None
· EcoR1
None
· Sterile DI water
To make final volume to 50ul
· Do not add any enzyme to the ‘-‘ tube
· Repeat with the other two plasmid samples
· Mix the tubes by flicking – DO NOT VORTEX
· Give a 5 second spin in the centrifuge to bring the contents to the bottom
· Incu.
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
Explain the basic mechanisms involved in DNA extraction.
Describe the steps involved in gDNA extraction from blood.
Explain the processes involved in quality and quantity check of extracted DNA using nanodrop technique.
Decribe the steps of quantity check of amplicon using flurometer.
Decribe the principle of dilution of amplicon.
Presented by,
Dr. Md. Mohiuddin Masum
Guided by,
Prof. Laila Anjuman Banu
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
Molecular Biology & Biotechnology(Practical) MANIKImran Nur Manik
a) Isolation of plasmid DNA
b) Estimation of DNA, RNA and oligonucleotides
c) Agarose-gel electrophoresis of nucleic acids
d) Determination of bacterial drug resistance by disk diffusion method.
e) Estimation of protein concentration by Lowry method
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
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.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
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https://www.etran.rs/2024/en/home-english/
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.
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In this book, we use conservation of energy techniques on a fluid element to derive the Modified Bernoulli equation of flow with viscous or friction effects. We derive the general equation of flow/ velocity and then from this we derive the Pouiselle flow equation, the transition flow equation and the turbulent flow equation. In the situations where there are no viscous effects , the equation reduces to the Bernoulli equation. From experimental results, we are able to include other terms in the Bernoulli equation. We also look at cases where pressure gradients exist. We use the Modified Bernoulli equation to derive equations of flow rate for pipes of different cross sectional areas connected together. We also extend our techniques of energy conservation to a sphere falling in a viscous medium under the effect of gravity. We demonstrate Stokes equation of terminal velocity and turbulent flow equation. We look at a way of calculating the time taken for a body to fall in a viscous medium. We also look at the general equation of terminal velocity.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
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3. Isolation and purification of genomic DNA from whole blood
Aim:
Introduction: Blood is a specialized body fluid composed of
cells suspended in a liquid called blood plasma. Whole blood
contains three types of cells: 1. Red blood cells (RBCs) 2. White
blood cells (WBCs) 3. Platelets Red blood cells (RBCs) do not
have any DNA, as they lose their nuclei during maturation. The
white blood cell (WBC) component of the blood contains the DNA.
The blood sample is treated with detergents, which break open the
cell membrane to release the contents. Enzymes are then used to
break down all the proteins, RNA, sugars and fats in the solution
4. Principle: Isolation and purification of high molecular
weight genomic DNA from fresh whole blood by using
highly efficient solution based system. Genomic DNA
purification from whole blood involves lysis of the red
blood cells with RBC Lysis Buffer followed by the lysis
of white blood cells and their nuclei with WBC Lysis
Buffer. Impurities like cellular 4 proteins are removed by
precipitation and short washing steps while high
molecular weight genomic DNA remains in the solution.
High quality genomic DNA is then purified by
isopropanol precipitation.
5. Kit Contents:
Materials Required But Not Provided: Glasswares: Conical flask, Measuring
cylinder, Beaker Reagents: Ethidium bromide (10 mg/ml), Ethanol, Distilled Water
Other requirements: 15 ml and 50 ml centrifuge tubes, Electrophoresis apparatus,
UV Transilluminator, Heating block or Water Bath, Vortex Mixer, Tabletop
Microcentrifuge (with rotor for 2.0 ml tubes), Micropipettes, Tips, Adhesive tape, Ice,
Microwave/Burner/Hotplate
6. Storage:Blood Genomic DNA Extraction Teaching
Kit (Solution Based) is stable for 6 months from the
date of manufacture without showing any reduction in
performance. On receipt, store the Control DNA at -
20o C and the 6X Gel Loading Buffer at 2-8o C. Other
reagents can be stored at room temperature (15-25o
C). WBC Lysis Buffer may form precipitate in cool
ambient conditions. In such condition, heat the bottle
before use at 55o C to dissolve the contents.
7. Procedure:
1. Take 300 μl of fresh whole blood in a 2.0 ml collection tube. Ensure that the blood sample is at
room temperature (15-25o C) before beginning the protocol.
2. RBC Lysis Add 900 μl of RBC Lysis Buffer and mix well by inverting the tube for 6-8 times.
Incubate at room temperature for 5 minutes. Mix the tube contents intermittently by inverting 2-3
times during incubation.
3. Centrifuge the tube at 15,000 rpm for 1 minute at room temperature. Discard the supernatant
carefully without disturbing the white pellet such that very small amount (~15 µl) of residue liquid
remains back in the tube.
4. Vortex the tube vigorously so as to resuspend the white blood cells completely.
5. WBC Lysis Add 300 μl of WBC Lysis Buffer to the resuspended white blood cells and gently
pipette to lyse the cells. Solution should become viscous. If any cell clumps are still present,
incubate the solution at 37o C (10 minutes) until the clumps dissolve. 6. Add 1.5 μl of RNase A
solution. Invert the tube 20-25 times to ensure thorough mixing of enzyme and incubate for 10
minutes at 37o C
8. 7. Cool the sample to room temperature before further processing.
8. Precipitation of Proteins Add 100 μl of Precipitation Buffer to the cell lysate.
Mix by vortexing for 30 seconds. Incubate on ice for 5 minutes, as some protein
clumps may be visible after vortexing.
9. Centrifuge at 14,000 rpm for 3 minutes at room temperature.
10. Precipitation of DNA Transfer the above supernatant to a new 2.0 ml
collection tube. Add 300 μl of 100% isopropanol and mix by inverting the tube
gently till the DNA in white fibrous form is visible (30-40 times).
11. Centrifuge at 15,000 rpm for 1 minute at room temperature. Small white
pellet of DNA will be visible. Discard the supernatant.
9. 12. Wash Remove the residual supernatant by carefully inverting the tube on
a clean tissue paper without disturbing the pellet. Add 300 μl of 70 % ethanol
to the DNA pellet and wash by inverting the tube 6- 8 times.
13. Centrifuge at 15,000 rpm for 2 minutes at room temperature. Carefully
discard the supernatant. The pellet may be very loose at this point, so the
supernatant should be carefully discarded without disturbing the pellet. Repeat
the wash step for one more time.
14. Invert the tube on a clean tissue paper and air-dry the pellet for 10-15
minutes.
15. DNA Elution Add 100 μl of Elution Buffer and vortex for 1 minute. Incubate
the tube at 65 C for 10 minutes to dissolve the DNA pellet completely
Storage OF DNA:Short Term 2-8 degrees
Long Term -20 Degrees
10. Agarose Gel Electrophoresis:
Step 1
• 1x TAE buffer is
prepared by adding
10ml of 50XTAE
buffer to 490ml of
sterile Dil.H20
Step 2
• To prepare 0.8%
agarose gel, add 0.4
agarose in 5oml of
1xTAE buffer.Heat then
cool down add
0.5moles Ethidium
Bromide and leave it for
30 mins to solidify
Step 3
• DNA sample(10ml) is
loaded with 2ml of 6x
gel loading buffer.Mix
them well by pipetting
and load samples
into the wells.
Step 4
• Electrophoresis:
• Connect the
power cord
• Red-Anode
• Black-Cathode
• 100-120 Volts.
• Depending on
the size of the
DNA to be
visualized.
11. Quantitation of DNA: Spectrophotometric analysis
and agarose gel electrophoresis will reveal the
concentration and the purity of the genomic DNA.
Absorbance readings at 260 nm should fall between
0.1 and 1.0. The 320 nm absorbance is used to
correct background absorbance. Purity is determined
by calculating the ratio of absorbance at 260 nm to
absorbance at 280 nm. The concentration of DNA is
calculated by the following formula: Concentration of
DNA sample (μg/ml) = 100 x A260 x dilution
factor
12.
13. Interpretation:
The lanes 1 and 2 demonstrate that highly purified blood genomic
DNA has been obtained with no visible RNA contamination when
electrophoresed on agarose gel. If RNA contamination is present , one would
see a faint and smeary RNA band below the genomic DNA as shown in lane 3
since RNA being of lower molecular weight than DNA runs faster than the
genomic DNA.RNA contamination is observed when the RNase treatment has
either been skipped or not been carried out properly.
An absorbance of 1.0 at 250 nm corresponds to approximately 50 µg/ml
of DNA .If the A260/A280 ratio , then the isolated DNA sample is considered to
be pure. If higher A260/A280 ratio is observed it indicates the possibility of
RNA contamination