The document describes the alkaline lysis method for plasmid purification from bacterial cells. It involves four main steps: 1) resuspending harvested cells, 2) lysing the cells with NaOH and SDS, 3) neutralizing with potassium acetate, and 4) clearing the lysate by centrifugation. This separates plasmid DNA from genomic DNA and other cell debris. The purified plasmid DNA can then be analyzed using agarose gel electrophoresis.
Presentation on nested pcr . contain types of pcr, protocol of nested pcr, advantages of nested pcr, disadvantages of nested pcr, application of nested pcr ,pictorial representation of pcr.
Reverse transcription of RNA, which refers to the conversion of the RNA template into its complimentary DNA strand (cDNA) is an essential step in the analysis of gene transcripts.
cDNA can be sequenced, cloned and applied to estimate the copy number of specific genes in order to characterize and to validate gene expression.
Molecular markers for measuring genetic diversity Zohaib HUSSAIN
Molecular markers for measuring genetic diversity
Introduction:
The molecular basis of the essential biological phenomena in plants is crucial for the effective conservation, management, and efficient utilization of plant genetic resources (PGR).
Determining genetic diversity can be based on morphological, biochemical, and molecular types of information. However, molecular markers have advantages over other kinds, where they show genetic differences on a more detailed level without interferences from environmental factors, and where they involve techniques that provide fast results detailing genetic diversity
Comparison of different methods
Morphological characterization does not require expensive technology but large tracts of land are often required for these experiments, making it possibly more expensive than molecular assessment. These traits are often susceptible to phenotypic plasticity; conversely, this allows assessment of diversity in the presence of environmental variation.
Biochemical analysis is based on the separation of proteins into specific banding patterns. It is a fast method which requires only small amounts of biological material. However, only a limited number of enzymes are available and thus, the resolution of diversity is limited.
Molecular analyses comprise a large variety of DNA molecular markers, which can be employed for analysis of variation. Different markers have different genetic qualities (they can be dominant or co-dominant, can amplify anonymous or characterized loci, can contain expressed or non-expressed sequences, etc.).
Genetic marker
The concept of genetic markers is not a new one; in the nineteenth century, Gregor Mendel employed phenotype-based genetic markers in his experiments. Later, phenotype-based genetic markers for Drosophila melanogaster led to the founding of the theory of genetic linkage. A genetic marker is an easily identifiable piece of genetic material, usually DNA that can be used in the laboratory to tell apart cells, individuals, populations, or species. The use of genetic markers begins with extracting proteins or chemicals (for biochemical markers) or DNA (for molecular markers) from tissues of the plant (for example, seeds, foliage, pollen, sometimes woody tissues).
Molecular markers In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA that is associated with a certain location within the genome. Molecular markers which detect variation at the DNA level such as nucleotide changes: deletion, duplication, inversion and/or insertion. Markers can exhibit two modes of inheritance, i.e. dominant/recessive or co-dominant. If the genetic pattern of homozygotes can be distinguished from that of heterozygotes, then a marker is said to be co-dominant. Generally co-dominant markers are more informative than the
Presentation on nested pcr . contain types of pcr, protocol of nested pcr, advantages of nested pcr, disadvantages of nested pcr, application of nested pcr ,pictorial representation of pcr.
Reverse transcription of RNA, which refers to the conversion of the RNA template into its complimentary DNA strand (cDNA) is an essential step in the analysis of gene transcripts.
cDNA can be sequenced, cloned and applied to estimate the copy number of specific genes in order to characterize and to validate gene expression.
Molecular markers for measuring genetic diversity Zohaib HUSSAIN
Molecular markers for measuring genetic diversity
Introduction:
The molecular basis of the essential biological phenomena in plants is crucial for the effective conservation, management, and efficient utilization of plant genetic resources (PGR).
Determining genetic diversity can be based on morphological, biochemical, and molecular types of information. However, molecular markers have advantages over other kinds, where they show genetic differences on a more detailed level without interferences from environmental factors, and where they involve techniques that provide fast results detailing genetic diversity
Comparison of different methods
Morphological characterization does not require expensive technology but large tracts of land are often required for these experiments, making it possibly more expensive than molecular assessment. These traits are often susceptible to phenotypic plasticity; conversely, this allows assessment of diversity in the presence of environmental variation.
Biochemical analysis is based on the separation of proteins into specific banding patterns. It is a fast method which requires only small amounts of biological material. However, only a limited number of enzymes are available and thus, the resolution of diversity is limited.
Molecular analyses comprise a large variety of DNA molecular markers, which can be employed for analysis of variation. Different markers have different genetic qualities (they can be dominant or co-dominant, can amplify anonymous or characterized loci, can contain expressed or non-expressed sequences, etc.).
Genetic marker
The concept of genetic markers is not a new one; in the nineteenth century, Gregor Mendel employed phenotype-based genetic markers in his experiments. Later, phenotype-based genetic markers for Drosophila melanogaster led to the founding of the theory of genetic linkage. A genetic marker is an easily identifiable piece of genetic material, usually DNA that can be used in the laboratory to tell apart cells, individuals, populations, or species. The use of genetic markers begins with extracting proteins or chemicals (for biochemical markers) or DNA (for molecular markers) from tissues of the plant (for example, seeds, foliage, pollen, sometimes woody tissues).
Molecular markers In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA that is associated with a certain location within the genome. Molecular markers which detect variation at the DNA level such as nucleotide changes: deletion, duplication, inversion and/or insertion. Markers can exhibit two modes of inheritance, i.e. dominant/recessive or co-dominant. If the genetic pattern of homozygotes can be distinguished from that of heterozygotes, then a marker is said to be co-dominant. Generally co-dominant markers are more informative than the
It is common for students to use kits without knowing exactly what the different solutions/buffers are doing or what are they composed of. This automate attitude is wrong, thus, a proper discussion over the ins and outs of DNA extraction kits is imperative. The Toxicologist Today gives a little help, if you know more help us by commenting.
L’électrophorèse en champ pulsé est le résultat de la combinaison d’une digestion par des enzymes de restriction à faible nombre de sites de coupure et d’une électrophorèse adaptée à la grande taille des produits de digestion. Le résultat est un profil de restriction intéressant tout le génome du microorganisme
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.
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.
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.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
2. PRINCIPLE Plasmid for routine molecular cloning method is often purified by one of the following methods :- Alkaline lysis method Boiling method Lithium chloride base The purity of plasmid isolated by these above methods depends on how efficiently a method can separate plasmid DNA from genomic DNA. Most of these plasmid purification methods allow the preferential recovery of circular plasmid DNA over linear chromosomal DNA. Sunday, April 04, 2010 2 HIMANSHU CHAUDHRY
3. ALKALINE LYSIS method Alkaline lysis method is one of the most commonly used method for lysis bacterial cells prior to plasmid purification. It has four basic steps :- 1. Resuspension : Harvested bacterial cells are resuspended by using solution I contains EDTA (ethylene diaminetetra-acetic acid) and Tris-CL. EDTA – chelates the magnesium and calcium ions Tris-CL – maintains pH. 2 . LYSIS : Cells are lysis with alkaline solution II contains NaOH and SDS (sodium dodecyl sulfate). NaOH -- denatures the chromosomal and plasmid DNAs as well as proteins. SDS -- solubilizes the phospholipids and protein components of the cell membrane, leading to lysis and release of the cell membrane. 3. NEUTRALIZATION : The lysate is neutralized by addition of solution III of acidic potassium acetate. The high salt concentration causes potassium dodecyl sulfate (KDS) to precipitate and denatured proteins, chromosomal DNA and cellular debris are co-precipitated in insoluble. . 4. CLEARING OF LYSATES : Precipitated debris is removed by either high speed centrifugation or filtration, producing cleared lysates Sunday, April 04, 2010 3 HIMANSHU CHAUDHRY
4. continue…. Step ‘s and procedure in ALKALINE LYSIS METHOD Sunday, April 04, 2010 4 HIMANSHU CHAUDHRY
5. . Continue….. Harvest cells by centrifugation Spin ~5,000 rcf Supernatant (clear) E. coli culture (cloudy) Pelleted cells Discard supernatant Residual media may interfere with downstream steps Resuspend cells in buffer Thoroughly resuspend cells, making sure that no clumps remain. P1 buffer contains: •Tris-Cl (buffering agent) •EDTA (metal chelator) •RNase A (degrades RNA)
6. Continue…. Sunday, April 04, 2010 6 Lyse cells with SDS/NaOH solution Adding buffer P2 causes solution to become viscous 1. Sodium dodecylsulfate • Dissolves membranes • Binds to and denatures proteins 2. NaOH • Denatures DNA Because plasmids are supercoiled, both DNA strands remain entangled after denaturation HIMANSHU CHAUDHRY
7. sodium dodecyl sulfate (SDS) potassium dodecyl sulfate (PDS) (H2O sol. = 10%) (H2O sol. < 0.02%) Continue… Neutralize NaOH with potassium acetate solution Mixing with buffer N3 causes a fluffy white precipitate to form. 1. Potassium acetate / acetic acid solution • Neutralizes NaOH (renatures plasmid DNA) • Converts soluble SDS to insoluble PDS 2. Guanidine hydrochloride (GuCl) • Chaotropic salt; facilitates DNA binding to silica in later steps Sunday, April 04, 2010 7 HIMANSHU CHAUDHRY
8. Continue…. Sunday, April 04, 2010 8 Separate plasmid DNA from contaminants by centrifugation Supernatant contains: - Plasmid DNA - Soluble cellular constituents Pellet contains: - PDS - Lipids - Proteins - Chromosomal DNA HIMANSHU CHAUDHRY
9. Sunday, April 04, 2010 9 Add cleared lysate to column and centrifuge The high ionic strength and presence of chaotropic salt causes DNA to bind to the silica membrane, while other contaminants pass through the column Centrifuge Nucleic acids Silica-gel membrane Flow through (discard) HIMANSHU CHAUDHRY
10. Sunday, April 04, 2010 10 Wash the silica membrane to remove residual contaminants Buffer PB contains isopropanol and GuCl Centrifuge PB buffer Nucleic acids Nucleic acids PB + contaminants Buffer PE contains ethanol and Tris-Cl Centrifuge PE buffer Nucleic acids Nucleic acids PE + contaminants (including residual GuCl) HIMANSHU CHAUDHRY
11. Sunday, April 04, 2010 11 Elute purified DNA from the column Buffer EB should be added directly to the membrane for optimal DNA recovery and to avoid possible EtOH contamination (from residual PE buffer) EB is 10 mM Tris-Cl (pH 8.5). TE or dH2O may also be used. Centrifuge EB buffer Nucleic acids EB + DNA HIMANSHU CHAUDHRY
12. Continue…. PLASMID PREPARATION 1.5ml of bacterial culture was taken in centrifuge tube ↓ Centrifuge of bacterial culture at 13,000 rpm ∕ 30 seconds ↓ Collection of pellet in fresh eppendorf’s tubes ↓ Addition of 100µl S1 buffer to the pellet ↓ Addition of 200µl S2 buffer and mixing of the sample by inverting 6-8 times ↓ Addition of 150µl of S3 buffer and mixing of the sample by inverting 6-8 times ↓ Addition of 450µl of P1 buffer and mixing of the sample by inverting 6-8 times ↓ Centrifugation at 13,000 rpm ∕ 30 sec ↓ Collection of supernatant in a fresh tube ↓ Addition of 20µl DBM into the supernatant and mixing the sample by inverting 6-8 times Sunday, April 04, 2010 12 HIMANSHU CHAUDHRY
13. Continue... Incubation at room temperature for 1 minute ↓ Centrifugation at 13,000 rpm ∕ 30 sec ↓ Removal of supernatant ↓ Addition of 500µl wash buffer to the pellet ↓ Centrifugation at 13,000 rpm / 30 sec ↓ Centrifugation until complete removal of wash buffer ↓ Addition of 20µl Elution buffer to the pellet ↓ Incubation at room temperature for 1 minute ↓ Centrifugation at 10,000 rpm/30 second ↓ Collection of Elutein a fresh tube ↓ store in −20°C in freeze Sunday, April 04, 2010 13 HIMANSHU CHAUDHRY
14. Gel electrophoresis Agarose gel electrophoresis is a widely used method that separates molecules based upon charge, size and shape. The purpose of the gel will be either to visualize the DNA,to quantify it or to isolate a particular band. Agarose forms a porous lattice in the buffer solution and the DNA must slip through the holes in the lattice in order to move toward the positive pole. DNA is visualized in the gel by addition of ethidium bromide , binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visible orange light. Sunday, April 04, 2010 14 HIMANSHU CHAUDHRY
15. PREP. Of 1% AGAROSE GEL Take one 250 mg Agarose tablet in 25 ml of 1x TAE buffer. The tablet gets disintegrated with in 1 min. Mix the content and heat it in a microwave for 30 seconds. Mix the content and allow the Agarose to cool to 50°C and pour it in a plate that was sealed on either sides using a tape. After the gel is solidified, remove the tape and use it for electrophoresis of DNA samples. Sunday, April 04, 2010 15 HIMANSHU CHAUDHRY
16. Continue…. Most Agarose gels: 1. 1% gels are common for many applications. 2. 0.7%: good separation or resolution of large 5–10kb DNA fragments 3. 2% good resolution for small 0.2–1kb fragments. 4. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Sunday, April 04, 2010 16 HIMANSHU CHAUDHRY
17. Continue… Buffer The most common buffers for Agarose gel: TAE: Tris acetate EDTA TBE: Tris/Borate/EDTA SB: Sodium borate. TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 17
18. Continue….. An agarose gel is prepared by combining agarose powder and a buffer solution. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 18 Buffer Flask for boiling Agarose
19. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 19 Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer. Buffer solution Agarose Powder
20. Melting the Agarose Agarose is insoluble at room temperature The agarose solution is boiled until clear Sunday, April 04, 2010 HIMANSHU CHAUDHRY 20
21. Gel casting tray & combs Sunday, April 04, 2010 HIMANSHU CHAUDHRY 21
22. Preparing the Casting Tray Sunday, April 04, 2010 HIMANSHU CHAUDHRY 22 Seal the edges of the casting tray and put in the combs. Place the casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray.
23. POURING THE GEL IN TRAY Sunday, April 04, 2010 HIMANSHU CHAUDHRY 23 Allow the agarose solution to cool slightly (~60°C) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.
24. Each of the gel combs should be submerged in the melted agarose solution. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 24
25. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 25 When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape.
26. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 26 Place the gel in the electrophoresis chamber.
27. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 27 DNA BUFFER WELLS ANODE (positive) CATHODE (Negative) Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.
28. Sample Preparation 6X Loading Buffer: Bromophenol Blue (for color) Glycerol (for weight) Sunday, April 04, 2010 HIMANSHU CHAUDHRY 28 Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.
29. Loading the Gel Sunday, April 04, 2010 HIMANSHU CHAUDHRY 29 Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.
30. Running the Gel Sunday, April 04, 2010 HIMANSHU CHAUDHRY 30 Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.
31. Sunday, April 04, 2010 HIMANSHU CHAUDHRY 31 Cathode (-) DNA (-) wells Bromophenol Blue Anode (+) After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.
32. Observe the gel under UV Light Sunday, April 04, 2010 HIMANSHU CHAUDHRY 32 Assessing your plasmid preparation 1. Quantify abundance (A260) and purity (A260/A280) 2. Verify by restriction digestion 3. Run undigested plasmid to see if it is mostly supercoiled ←SUPERCOILED ←DENATURED