This is a descriptive and simple molecular biology lab manual.For students who do not have P.C in their home and can not use the virtual lab videos available online.
Expt. 2 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
What is pyrogens?
Sources of pyrogens and its elimination methods
Tests for pyrogens-
1. In Vitro Test / LAL Test
2. In Vivo Test / Rabbit Test.
Objective
Principle
Requirements
Procedure
Observation table
Result and interpretation
Expt. 7 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of frog ringer solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
Expt. 2 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
What is pyrogens?
Sources of pyrogens and its elimination methods
Tests for pyrogens-
1. In Vitro Test / LAL Test
2. In Vivo Test / Rabbit Test.
Objective
Principle
Requirements
Procedure
Observation table
Result and interpretation
Expt. 7 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of frog ringer solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
DOI: 10.21276/ijlssr.2016.2.3.16
ABSTRACT- The present research article was described about the hypotriglycerdemic activity of Withania coagulans
bud extract. Withania coagulans Dunal belonging to the family Solanaceae is a small bush which is widely spread in
South Asia. The biological activity of with anolides from Withania coagulans has antihyperglycaemic activity and the
plant is commonly called as Indian cheese maker due to the milk coagulation characteristics of the bud. The present study
was to investigate preliminary studies shows satisfactory result. The chromatographic studies like TLC, HPTLC and
HPLC show good spot. HPTLC shows maximum height and area of 18.83%.HPLC shows maximum peak at 1.867
minutes having area coverage of 87.4%.The free radical scavenging activity of chloroform fraction (CF) of a crude drug
shows 510μg/ml of scavenging activity. The IC50 value for MTT assay was found to be 84.7μg/ml. The GLUT4 study
shows significant uptake of glucose. PPAR gamma activity regulation of glucose disposal and insulin sensitivity in the
skeletal muscles shows concentration dependence response using standard Pioglitazone. The bud of Withania coagulants
will be a promising medicine for more ailments.
Key-words- Withania coagulants, Hypotriglycerdemic, HPLC, HPTLC, GLUT-4, MTT assay
Field of pharmacology
Pharmacology practice school report .
Final year b pharmcy
Domain-Pharmacology
It include
1) experimental pharmacology
2) Toxicity study
3)pharmacovigilance
Biochemistry is a basic science which deals with chemical nature and chemical behaviour of living matter and with the reactions and processes they undergo.
Biochemistry involves the study of:
Chemical constituents of living matter.
Chemical changes which occur in the organism during digestion, absorption and excretion.
Chemical changes which occur during growth and multiplication of the organism.
Transformation of one form of chemical constituent to the other.
Energy changes involved in such transformation.
Note:- The term “Biochemistry” was first introduced by German chemist Carl Neuberg in 1903 from Greek word “bios” means “life”.
It is mainly deals with the biochemical aspects that are involved in several conditions.
The results of qualitative and quantitative analysis of body fluids assist the clinicians in the diagnosis, treatment and prevention of the disease and drug monitoring, tissue and organ transplantation, forensic investigations and so on.
Various biological fluids subjected to chemical tests and assays include blood, plasma, serum, urine, cerebrospinal fluid (CSF), ascetic fluid, pleural fluid, faeces, calculi and tissues.
Note:- Modern day medical practice is highly dependent on the laboratory analysis of body fluids, especially the blood. The disease manifestations are reflected in the composition of blood and other tissues.
Hence, the demarcation of abnormal from normal constituents of the body is another aim of the study of clinical biochemistry.
Experiment 1 Introduction to In-Vitro pharmacology and physiological salt so...Kanchan Chouksey
In Vitro pharmacology studies the biological effects of a drug in an isolated environment, such as cell lines or tissues.
This setup conveniently eliminates whole organism physiological influences allowing for a detailed analysis a compound’s impact.
Heart is supplied by autonomic nervous system.
Adrenaline acts as agonist.
It acts on β-receptors and increases heart rate and amplitude.
Acetylcholine acts on muscarinic receptors as an agonist and decreases the heart rate and amplitude.
Excess concentration of KCl stops the heart beat during diastolic phase.
Ca2+ excess concentration stops the heart beat during systolic phase.
K+ and Ca2+act on cardiac muscle through non-receptor mechanism of action.
This is a lecture presented by Dr.Omer Yahia Describing the first step of in the Role of molecular diagnostics through out the life. Give a brief shading out on the procedures for sample collection and types of diseases and syndromes undergone such tests .
Basic Mutagenic signal Transduction or the cancer signal transduction that control cell cycle are important pathways to understand cancer in molecular level and to invent targeted treatment.
These are Lectures of Basic molecular pharmacology presented by Dr.Omer Yahia In coordination with faculty of pharmacy university of Khartoum, al-Neelen medical research center, GENOM Professional training center and National center of Research (Ministry of science and communication).
Vitiligo is an acquired organ specific autoimmune disease of unknown etiology characterized by white patches in the skin. The patho-physiology of this disease is characterized by loss of functional melanocytes associated with infiltration of reactive T cells and dendritic cells. So, there are many evidences support that autoimmunity has a great role in Vitiligo-pathogenesis. Many efforts were made in areas of Histopathology, Immunology, and molecular biology to solve vitiligo puzzle. However, no clear etiology was described. We tried here to review some histopathological findings that make strong evidences for the autoimmunity in this disease.
Normal Labour/ Stages of Labour/ Mechanism of LabourWasim Ak
Normal labor is also termed spontaneous labor, defined as the natural physiological process through which the fetus, placenta, and membranes are expelled from the uterus through the birth canal at term (37 to 42 weeks
Francesca Gottschalk - How can education support child empowerment.pptxEduSkills OECD
Francesca Gottschalk from the OECD’s Centre for Educational Research and Innovation presents at the Ask an Expert Webinar: How can education support child empowerment?
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Embracing GenAI - A Strategic ImperativePeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
4. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل4
Diagram describes the role of diagnosis across the continuum of health
care:
Diagram describes the role of molecular diagnostics across the
continuum of health care:
Reference: Introduction to molecular diagnosis (the essential of diagnostic series), AdvaMedDx
and Dx Insights
5. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل5
الوعول هكونبث:
Molecular diagnostic and research lab consist of 5 rooms and office + class
room.
1 .Sample preparation
room.
2. Reagent preparation
room.
3. PCR room.
4. Gell electrophoresis
room.
5. UV.room
6. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل6
الوعوليت الوخبطر هن الوقبيت
ًالوو:ال انًٕادكيًيبئيتFirst: the chemicals:
ِانخطٕس ٍي ػبنيت ّدسخ ػهي ّاندضيئي األحيبء يؼًم في انًسخخذيت انكيًيبئيت انًٕاد ٍي انؼذيذ.انششكبث ٔحمٕو
إ ّحُب ػببساث ٔأ ػاليبث بٕػغ ّانًظُؼيؼشف فيًب ّٔاػح بظٕس ّانكيًيبئي انًٕاد ِْز خطٕسة نيMSDS
Material Safety Data Sheet)(.ّانًخبؽشانظحي بيبَبث ، انكيًيبئي اإلسى ػهي ححخٕي انًؼهٕيبث ِْز
ّاطبب ٔلٕع حبنت في ّانالصي ّاألٔني ٔاإلسؼبفبث.ٌبيب ،اإلَفدبس ٔا انحشيك يخبؽش ٔ ،ّانفيضيبئي انخٕاص أيؼباث
ِنهًبد ٍاألي اإلسخخذاو ؽشيمت ٔ ،ِانخطش انخفبػألث.
The following chemicals are particularly noteworthy:
1. Phenol and Chloroform:
Can cause severe burns so use protective measures whenever
appropriate. Wear gloves when dealing with these chemicals and use
the laminar hood in case of Phenol.
2. Acrylamide :
Potential neurotoxin, avoid inhalation by using face masks.
3. Ethidium bromide :
Carcinogen, use protective gloves in case of working in an Ethidium bromide
contaminated zone in the Lab.
NOTES:
These chemicals are not harmful if used properly.
Always wear gloves when using potentially hazardous chemicals and never
mouth-pipet them.
If you accidently splash any of these chemicals on your skin, immediately
rinse the area thoroughly with water and inform the instructor.
Discard the waste in appropriate container.
Second: Ultraviolet Light (in normal PCR not real time):
Exposure to ultraviolet light can cause acute eyes irritation. Since the retina can’t detect
UV light, you can have serious damage and not realize it until 30 min to 24 hours after
exposure. Therefore, always wear appropriate eye protection when using UV lamps.
Third: Electricity:
The voltages used for electrophoresis are sufficient to cause electrocution. Cover the
buffer reservoirs during electrophoresis. Always turn off the power supply and unplug the
leads before removing a gel.
7. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل7
Safety roles:
1. Place bags, Lab coats, books…. etc. in specific locations
(NEVER ON THE BENCH TOPS).
2. No eating or drinking in the laboratory. Do not store food in the laboratory.
3. No pipetting by mouth. Use mechanical pipetting device only.
4. Wear lab coats, disposable gloves, and safety glasses when appropriate.
5. Keep all noxious and volatile compounds in the fume hood.
6. Dispose of all biological waste into appropriate receptacles. Live cultures can
be treated with Clorox bleach or autoclaved. Do not toss out into regular trash
or down drains without autoclaving.
7. Do not use plastic or polycarbonate containers, tests tubes, pipettes etc. with
phenol and chloroform. Instead use polypropylene or glass with these organic
compounds.
8. Do not dispose of hazardous or noxious chemicals in laboratory sink. Use
proper containers in fume hood.
9. Wash your hand before leaving the Lab.
10. Report all accident to Lab manager immediately.
11. General housekeeping :
All common area should be kept free of clutter and all dirty dishes,
electrophoresis equipment, etc. should be dealt with appropriately.
Since you will use common facilities, all solutions and everything stored
in an incubator, refrigerator, etc. must be labeled. In order to limit
confusion.
Unlabelled material found in refrigerators, incubators, or freezers may be
destroyed.
Always mark the backs of the plates with your initials, the date, and
relevant experimental data, e.g. type of tissue and test.
8. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل8
Reagent preparation:
General information:
Molar solutions:
A molar solution is one which 1 liter of solution contains the number of grams equal to
its molecular weight.
E.g. to make up 100 ml of a 5M NACL solution = 58.456 (mwt of NACL) g/mol x 0.1
liter = 29.29 g in 100 ml of solution
Percent solutions (%):
Percentage (w/v) = weight (g) in 100 ml of solution;
Percentage (v/v) = volume (ml) in 100 ml of solution
To make 100 ml of TE buffer (10 mM Tris, 1 mM EDTA), combine 1 ml of a 1 M Tris
solution and 0.2 ml of 0.5 M EDTA and 98.8 ml sterile water. The following is useful for
calculating amounts of stock solution needed: C i x V i = C f x V f , where C i = initial
concentration, or conc of stock solution; V i = initial vol, or amount of stock solution
needed C f = final concentration, or conc of desired solution; V f = final vol, or volume
of desired solution
E.g. to make a 0.7% solution of Agarose in TBE buffer, weight 0.7 of Agarose and bring
up volume to 100 ml with TBE buffer.
X solutions:
Many enzymes buffers are prepared as concentrated solutions,
E.g. 5X or 10X (5 or 10 times the concentration of the working solution) and are diluted
such that the final concentration of the buffer in the reaction is 1X.
E.g. to set up a restriction digestion in 25 micro liter, one would add 2.5 micro.l of a 10 x
buffer, the other reaction components, and water to final volume of 25 micro.l
9. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل9
DNA extraction methods
In this Experiment you need to isolate some DNA from a human test subject
Why, you may ask, do you need a human DNA?
Scientists isolate DNA for variety of reasons, some of which
include:
Genetic testing.
Body identification.
Analysis of forensic evidence.
DNA extraction is typically the first step in longer process.
DNA extraction is an important part of that process because the
DNA first needs to be purified away from proteins and other cellular contaminants.
We need cells, because that’s where the DNA is. Inside almost every cell in our bodies is
nucleus, and inside each nucleus is about two meters of DNA.
In diagnostic kits, they always supply all reagents needed for DNA or RNA extraction
and also their own protocol to achieve the highest concentration. But these are general
methods for extraction in case we have no Kits.
High salt extraction methods:
Materials:
Biological safety hood” exclusively used for nucleic acid extraction.
Warm water path.
Micro centrifuge.
Micropipettors.
Eppendorff tubes.
Tips.
vortex
Lysis buffer.
High salt solution NaCl 6 M.
Isopropyl alcohol.
Ethanol.
Chloroform.
The steps you will follow to purify DNA from a cheek swab,
blood sample or cancer cells are shown below:
1. Collect cheek cells, serum or minced cancer cell.
10. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل10
2. Burst cells open to release DNA.
3. Separate DNA from proteins and other debris.
4. Isolate concentrated DNA.
The procedures:
1. Using the micropipettor, add 100 ul lysis solution to
the Eppendorff tube and vortex for 2 min.
2. Next you will place the sample 100ul into the
Eppendorff tube.
3. Place the tube into the warm water 550
C for 2 hours.
The solution you just added contains two important
ingredients, Detergent and enzyme called proteinase
K. The detergent disrupts the cell membrane and
nuclear envelope, causing the cells to burst open and
release their DNA. The DNA is still wrapped very
tightly around proteins called histones; proteinase K
cuts apart the histones to free the DNA.
The cells have stayed in the warm water bath long
enough for the DNA to be freed from the cells, and we
have removed the swab from the tube.
4. Add 160ul of concentrated salt solution to your tube.
The salt causes protein and other cellular debris to
clump together.
5. Add 240ul chloroform to support precipitation of
protein and cellular debris.
6. Shake the tube vigorously by a shaker for 20 min to
make sure that chloroform collect all debris and
protein.
7. Place the tube into the centrifuge. In order to balance
the centrifuge, a tube containing water is placed
opposite your tube click the lid to close the centrifuge
and turn it on at 8000 rpm for 15 min.
Inside the centrifuge, the tubes spin around at high
speed. The heavy clumps of protein and cellular debris
to sink to the bottom of the tube, while the strands of
DNA remain distributed through the liquid. Then use
the micropipettor to carefully remove the top aqueous
liquid (which contains DNA) and place it into a clean
tube. The proteins and other cellular debris stay in the
chloroform layer.
11. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل11
8. After that add 240 ul isopropyl alcohol to the tube. Then mix
the isopropyl alcohol into the DNA by inverting it several
times. Because DNA is not soluble (does not stay dissolved)
in isopropyl alcohol, it comes out of solution. You can now
see the clumped DNA with your naked eye. Then Place the
tube into the centrifuge and click on the lid to close it and
turn it on. This time after the sample spins in the centrifuge
800 rpm for 5 min, the DNA sinks to the bottom of the tube
then discard isopropyl alcohol carefully.
9. Add 250 ul ethanol 70% and vortex the sample for 2 min
then centrifuge at high speed for 2 min then discard ethanol
carefully.
10. Once the liquid is removed and the DNA is allowed to dry,
you can re-dissolve it in the solution of your choice. You
can store it in the freezer for many years, or you can move
on to your next experiment. YOU HAVE JUST EXTRACTED DNA.
RNA extraction
Preparation of the sample and total RNA extraction
Diagnostic sample to be submitted for RNA extraction need to be properly prepared.
Materials:
“biological safety hood” exclusively used for nucleic acid extraction.
Pipettes.
Disinfectant solution.
Diagnostic samples.
Mencer.
Micro centrifuge.
vortex
Falcon and Eppendorff Tubes.
Eppendorff tubes with lysis buffer for RNA extraction.
1. Write the identification number of the related sample on
each tube with a permanent marker.
2. Using a sterile scissor or surgical blades, cut small blocks
of tissue from organ under examination and mince it
mincer.
3. Finally collect the homogenate in a tube.
12. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل12
Total RNA extraction:
1. Before you added the lysis buffer the sample is still
dangerous especially that containing viruses. A filter face
mask can also be worn to increase operator protection.
2. In diagnostic laboratory RNA extraction is commonly
performed using commercial kits. Commercial RNA
extraction kits are generally based on the same principles
and should be used following the manufacturer’s
instructions.
3. Begin the working session by marking the tube the
corresponding sample identification number.
4. Extraction procedure started with the lyses of the tissues
in order to free the nucleic acid from the cells. This is done
by adding the sample to the lysis buffer provided in the kit
according to the proportion recommended by the
manufacturer. This buffer contains reagents such as
guanidine and Tripon X that destroy cells, denature
protein, and inactivate RNases.
5. Dispensed the required volume of the lysis buffer into each
tube remembering that guanidine is harmful by inhalation,
in contact with skin and if swallowed.
6. Addition of the sample is now required, note that for each
patch of sample a negative control should be included by
adding distilled water to the lysis buffer instead of the
sample.
7. The correct amount of the homogenized tissue under
examination is now added to the lysis buffer. From now on
the operator can continue to work without face mask as
even virus containing sample is no longer infectious.
8. Vortex the solution in order to homogenized reagents and to
avoid pelleting of any precipitate in the tube.
9. The correct amount of the homogenized tissue under
examination is now added to the lysis buffer. From now on
the operator can continue to work without face mask as even
virus containing sample is no longer infectious.
10. Vortex the solution in order to homogenized reagents and to
avoid pelleting of any precipitate in the tube.
11. Loaded a lysate on a silica membrane column provided which
adsorb nucleic acid.
12. Identify each column with the identification number of the
13. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل13
sample.
13. Dispense the lysate in to the middle of the column
making sure that the filter are not touch by the tips in
order to avoid breaking of the glass fiber.
14. Since this is a high risk contamination procedure, it is
strongly recommended that one tube is opened at a
time and to change tip after each sample in order to
avoid transfer of RNA by contaminated tips.
15. Remember to change gloves before leaving the hood.
16. Centrifugation is now to performed to allow adsorption
of nucleic acids to the filter.
17. Now return to the hood and prepare new collection
tubes into which the column will be placed for the next step of the
procedure.
18. Discard the flow-through into the waste vessel. During this step
total DNA present in the sample is degraded and only the total
RNA of the sample remains on the filter.
19. Additional washing and drying steps are now performed using
washing buffers specifically developed to remove salts
metabolites and macromolecular cellular components from the
RNA sample. Remember that all these washing reagents contain
guanidine which is harmful by inhalation, in contact with skin and
if swallowed.
20. Centrifuge and then after washing, RNA elution is now possible. Prepare
nuclease-free collection tubes that have been properly identified in order to collect
RNA elute and to store it.
21. Elution of RNA is done by adding RNase free water. The water dissolves the
weak ionic binding between the silica membrane and the nucleic acid.
22. After a final centrifugation, the RNA is eluted and the extraction is completed and
the total RNA in the sample is collected in the tube.
23. To ensure RNA stability store RNA at -70 o
C.
14. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل14
Agarose gel electrophoresis (basic method)
You are holding a small plastic tube with some clear liquid in it.
You've been told that the liquid contains DNA strands of several
different lengths
Your job is to figure out what those lengths are.
The DNA strands are molecules so tiny that you can't see them
even under most microscopes. Is there a way to sort and
measure the DNA strands in your tube even though you can't see
or touch them?
There is! It's called gel electrophoresis.
Scientists use gel electrophoresis whenever they need to sort
DNA strands according to length. This technique is also useful
for separating other types of molecules, like proteins.
Equipments:
Here is what you will need to make a gel:
1: Powdered agarose
2: TBE 1% buffer 8 PH.
3: A flask.
4: A microwave.
5: The gel mold and the gel comb.
15. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل15
Proceduer:
Follow along with the STEPS shown down!
STEP 1: Make the gel
STEP 2: Set up the gel apparatus
STEP 3: Load the DNA sample into the gel
STEP 4: Hook up the electrical current and run the gel
STEP 5: Stain the gel and analyze the results
The "gel" is the filter that sorts the DNA strands.
It's like sponge mode of Jell With many small
holes in it.
We place DNA samples into holes at one end of
the gel.
"Electrophoresis" is how we push the DNA strands
through the gel filter.
By adding an electrical current, we can make the
DNA move.
Short strands move through the holes than long
strands. Over time, the shorter strands in the sample
will move farther away from the starting point than
the longer strands of the same length will move at
the same speed and end up grouped together. In this
way, the DNA strands in the sample sort
themselves.
Staining the sorted groups of DNA makes them
visible to the naked eye.
Although we can't see a single DNA strand, we can
see large groups of stained DNA strands.
1. Put a small amount 2 g of agarose into the flask.
Agarose is a dried powder similar to gelatin but
mode from seaweed.
2. Add 200 ml liquid TBE buffer 1% PH 8 to the
flask.
The buffer is salt water solution consist of EDTA+
Boric Acid + Tris hydrochloride dissolved in
distilled water that will let electrical charges flow
through the gel.
16. .
ّانضأي ّانطبي انخمُيت كهيت
الجزيئي التشخيص معمل16
3. We've loosely placed plastic wrap over
the top of the flask to prevent the liquid
from boiling.
4. Place the flask containing the buffer and
agarose mixture inside the microwave.
Heat the mixture until the agarose melts
into the buffer.
5. We've removed the plastic wrap from
the top of the flask and pour the melted
agarose mixture into the mold. Notice
that the mold has rape on each end to
hold in the melted agarose.
6. Place the comb into the gel on one end.
7. The notches in the gel mold hold it in
place.
8. Let the gel cool and solidify. This usually
takes about half an hour, but we’ll speed
up the clock for you. As gel cools, tiny
holes will form in it.
9. Carefully remove the comb, leaving
empty wells for DNA samples. Your gel
is now ready to run.
10. Now it's time to set up the electrophoresis
box. You'll need the gel you just made, an
electrophoresis box and another bottle of
TBE 1% buffer.
11. Pour the buffer into electrophoresis box.
12. Place the gel, still in the mold, into the
electrophoresis box. We have removed the tape from
the ends of the gel mold. The gel should be just
barely submerged in the buffer.
13. The buffer conducts the electrical current from one
end of the gel to the other. It'll also keep the gel
from drying out during the experiment.
14. You're ready to load the DNA sample into the gel.
Here's what you'll need:
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Equipment:
Loading buffer (bromophenol blue dye).
DNA samples.
DNA size standard (Ladder).
Micropipette.
Gel box with buffer and gel.
Pipette tips.
15. With a clean pipette tip, use the micropipettor
to suck up some loading buffer, and then add
it to the DNA sample. DNA samples are
prepared in clear liquid solution that would be
hard to see if you tried to load it directly into a
well. The loading buffer contains
Bromophenol Blue that makes the sample
easy to see. It's also slightly goopy. This
makes the DNA sample thicker, so that it will
drop into the well instead of floating away.
The DNA size standard already contains
loading buffer.
16. Next, you will use micropipettor to transfer
the DNA sample from the tube into the well of
the gel. First, suck up some of the DNA
sample into pipet tip.
17. Eject the DNA sample from the pipet into the
first well of the gel.
18. Loading the sample into the wells takes some
practice, so don’t be disappointed if you miss
your target the first few times.
19. Using a clean pipet tip, use the micropipettor to
suck up some DNA size standard.
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20. Transfer the DNA size standard into the next empty well. The DNA size standard
contains DNA strands of known lengths. Running it on the gel will give you a
reference by which to estimate the lengths of DNA strands in your sample.
21. It's time to turn on the electrically and run your gel!. When you turn on the power,
the black end will generate a negative charge. The red end will generate a positive
charge. Together, they will pass the current through the gel 200 Volt and 100
mAmp for 3 Hours. DNA has a negative charge. To move the DNA through the
gel, you must put the black cord – the negative charge – closest to wells!
22. Plug the black cord from the electrophoresis box into the matching outlet on the
power supply. Click the location on the power supply where you want to plug in
the black cord. And do the same with the red cord then turn on the power supply.
23. Your gel is off and running! Let's look into the gel box to see what's
happening.Check for tiny air bubbles coming of electrodes at both ends of the
electrophoresis box. These bubbles are your proof that a current is running.
Repelled by the negative charge, the DNA moves through the gel toward the
positive charge at the other end. Short DNA strands move through the holes in
the gel more quickly than long strands. Over time, the shorter DNA strands
will migrate farther from the starting point than the longer strands. We can't
actually see the migrating DNA bands (grey), but we can see the blue dye
from the loading buffer as it migrates.
24. You've finished running your gel. We've taken the gel mold with the gel in it out
of the electrophoresis box.
25. First, you need to stain the DNA in your gel using DNA staining solution. The
stain is a chemical called Ethidium bromide 10 mg/ml, which binds to DNA and
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shows up under fluorescent light.
Although we can't see single DNA
strands, we can see large groups of
stained DNA strands. These groups
will show up as bands in the gel.
26. Drag the gel out of the mold and put
it into the DNA staining solution.
Because Ethidium bromide binds to
DNA, it can damage the DNA in your
cells. If you stain a gel in real life,
wear gloves and avoid direct contact
with the staining solution.
27. It takes about half an hour to the
DNA in the gel. We'll speed up the
clock for you.
28. Remove the gel from the staining
solution and place it in the UV
(ultraviolet) light box.
29. Turn on the UV box.
30. Ultraviolet (UV) light from the box can
damage your eyes, If you do this in real life,
be sure to wear protective goggles.
31. Now you can determine the approximate
lengths of the DNA strands in your sample!
Compare the bands from the DNA sample
with the bands of known length from the DNA
size standard. Write the estimated length, in
base pairs (bp), for each band in your DNA
sample. Electrophoresis cannot tell us the
exact DNA lengths of DNA strands, just a
good estimation. So, give it your best guess
based on the DNA size standards.
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TBE preparation:
TBE stands for Tris Borate EDTA.
People also use TAE (Tris Acetate EDTA). Make up a 10x stock using cheap reagents.
Do not use expensive 'analytical grade' reagents. Cheap Tris base and boric acid can be
bought in bulk.
Recipe for 2L of 10xTBE
218 g Tris base
110 g Boric acid
9.3 g EDTA
Dissolve the ingredients in 1.9 L of distilled water. pH to about 8.3 using NaOH and
make up to 2 L.
Thermal Cycling Profile for Standard PCR
Initial denaturation:
It is very important to denature the template DNA completely. Initial heating of the PCR
mixture for 2 minutes at 94°–95°C is enough to completely denature complex genomic
DNA so that the primers can anneal to the template as the reaction mix is cooled. If the
template DNA is only partially denatured, it will tend to “snap-back” very quickly,
preventing efficient primer annealing and extension, or leading to “self-priming,” which
can lead to false-positive results.
Denaturation step during cycling:
Denaturation at 94°–95°C for 20–30 seconds is usually sufficient, but this must be
adapted for the thermal cycler and tubes being used. (For example, longer times are
required for denaturation in 500 µl tubes than in 200 µl tubes.) If the denaturation
temperature is too low, the incompletely melted DNA “snaps-back” as described earlier,
thus giving no access to the primers. Use a longer denaturation time or higher denaturing
temperature for GC-rich template DNA.
Note: Never use a longer denaturation time than absolutely required for complete
denaturation of template DNA. Unnecessarily long denaturation times decrease the
activity of Taq DNA Polymerase.
Primer annealing:
For most purposes, annealing temperature has to be optimized empirically. The choice of
the primer annealing temperature is probably the most critical factor in designing a high
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specificity PCR. If the temperature is too high, no annealing occurs, but if it is too low,
non-specific annealing will increase dramatically. Primer-dimers will form if the primers
have one or more complementary bases so that base pairing between the 3' ends of the
two primers can occur.
Primer extension:
For fragments up to 3 kb, primer extension is normally carried out at 72°C. Taq DNA
Polymerase can add approximately 60 bases per second at 72°C. A 45-second extension
is sufficient for fragments up to 1 kb. For extension of fragments up to 3 kb, allow about
45 seconds per kb. However, these times may need to be adjusted for specific templates.
For improved yield, use the cycle extension feature of the thermal cycler. For instance,
perform the first 10 cycles at a constant extension time (e.g. 45 s for a 1 kb product).
Then, for the next 20 cycles, increase the extension time by 2–5 s per cycle (e.g. 50 s for
cycle 11, 55 s for cycle 12, etc.). Cycle extension allows the enzyme more time to do its
job, because as PCR progresses, there is more template to amplify and less enzyme (due
to denaturation during the prolonged high PCR temperatures) to do the extension.
Cycle number:
In an optimal reaction, less than 10 template molecules can be amplified in less than 40
cycles to a product that is easily detectable on a gel stained with Ethidium bromide. Most
PCRs should, therefore, include only 25 to 35 cycles. As cycle number increases,
nonspecific products can accumulate.
Final extension:
Usually, after the last cycle, the reaction tubes are held at 72°C for 5–15 minutes to
promote completion of partial extension products and annealing of single-stranded
complementary products.