DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid.- [https://www.khanacademy.org/science/...dna.../dna-cloning.../a/overview-dna-cloning]
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Genetic engineering is the process of manually adding new DNA to an organism. The goal is to add one or more new traits that are not already found in that organism. Examples of genetically engineered (transgenic) organisms currently on the market include plants with resistance to some insects, plants that can tolerate herbicides, and crops with modified oil content
description of plasmids and types and importance of plasmids and artificial plasmids(PBR322,cosmids,phagemids) and selection of the recombinants and uses and advantages and disadvantages of the plasmids
“The technique in which a DNA fragment carrying a gene inserted into the cloning vector, this vector is introduced in the living cell and subsequent propagation of this recombination DNA molecule into many copies is known as gene cloning.
DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid.- [https://www.khanacademy.org/science/...dna.../dna-cloning.../a/overview-dna-cloning]
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Genetic engineering is the process of manually adding new DNA to an organism. The goal is to add one or more new traits that are not already found in that organism. Examples of genetically engineered (transgenic) organisms currently on the market include plants with resistance to some insects, plants that can tolerate herbicides, and crops with modified oil content
description of plasmids and types and importance of plasmids and artificial plasmids(PBR322,cosmids,phagemids) and selection of the recombinants and uses and advantages and disadvantages of the plasmids
“The technique in which a DNA fragment carrying a gene inserted into the cloning vector, this vector is introduced in the living cell and subsequent propagation of this recombination DNA molecule into many copies is known as gene cloning.
R3 Stem Cells and Kidney Repair A New Horizon in Nephrology.pptxR3 Stem Cell
R3 Stem Cells and Kidney Repair: A New Horizon in Nephrology" explores groundbreaking advancements in the use of R3 stem cells for kidney disease treatment. This insightful piece delves into the potential of these cells to regenerate damaged kidney tissue, offering new hope for patients and reshaping the future of nephrology.
Global launch of the Healthy Ageing and Prevention Index 2nd wave – alongside...ILC- UK
The Healthy Ageing and Prevention Index is an online tool created by ILC that ranks countries on six metrics including, life span, health span, work span, income, environmental performance, and happiness. The Index helps us understand how well countries have adapted to longevity and inform decision makers on what must be done to maximise the economic benefits that comes with living well for longer.
Alongside the 77th World Health Assembly in Geneva on 28 May 2024, we launched the second version of our Index, allowing us to track progress and give new insights into what needs to be done to keep populations healthier for longer.
The speakers included:
Professor Orazio Schillaci, Minister of Health, Italy
Dr Hans Groth, Chairman of the Board, World Demographic & Ageing Forum
Professor Ilona Kickbusch, Founder and Chair, Global Health Centre, Geneva Graduate Institute and co-chair, World Health Summit Council
Dr Natasha Azzopardi Muscat, Director, Country Health Policies and Systems Division, World Health Organisation EURO
Dr Marta Lomazzi, Executive Manager, World Federation of Public Health Associations
Dr Shyam Bishen, Head, Centre for Health and Healthcare and Member of the Executive Committee, World Economic Forum
Dr Karin Tegmark Wisell, Director General, Public Health Agency of Sweden
Telehealth Psychology Building Trust with Clients.pptxThe Harvest Clinic
Telehealth psychology is a digital approach that offers psychological services and mental health care to clients remotely, using technologies like video conferencing, phone calls, text messaging, and mobile apps for communication.
Health Education on prevention of hypertensionRadhika kulvi
Hypertension is a chronic condition of concern due to its role in the causation of coronary heart diseases. Hypertension is a worldwide epidemic and important risk factor for coronary artery disease, stroke and renal diseases. Blood pressure is the force exerted by the blood against the walls of the blood vessels and is sufficient to maintain tissue perfusion during activity and rest. Hypertension is sustained elevation of BP. In adults, HTN exists when systolic blood pressure is equal to or greater than 140mmHg or diastolic BP is equal to or greater than 90mmHg. The
ICH Guidelines for Pharmacovigilance.pdfNEHA GUPTA
The "ICH Guidelines for Pharmacovigilance" PDF provides a comprehensive overview of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines related to pharmacovigilance. These guidelines aim to ensure that drugs are safe and effective for patients by monitoring and assessing adverse effects, ensuring proper reporting systems, and improving risk management practices. The document is essential for professionals in the pharmaceutical industry, regulatory authorities, and healthcare providers, offering detailed procedures and standards for pharmacovigilance activities to enhance drug safety and protect public health.
CRISPR-Cas9, a revolutionary gene-editing tool, holds immense potential to reshape medicine, agriculture, and our understanding of life. But like any powerful tool, it comes with ethical considerations.
Unveiling CRISPR: This naturally occurring bacterial defense system (crRNA & Cas9 protein) fights viruses. Scientists repurposed it for precise gene editing (correction, deletion, insertion) by targeting specific DNA sequences.
The Promise: CRISPR offers exciting possibilities:
Gene Therapy: Correcting genetic diseases like cystic fibrosis.
Agriculture: Engineering crops resistant to pests and harsh environments.
Research: Studying gene function to unlock new knowledge.
The Peril: Ethical concerns demand attention:
Off-target Effects: Unintended DNA edits can have unforeseen consequences.
Eugenics: Misusing CRISPR for designer babies raises social and ethical questions.
Equity: High costs could limit access to this potentially life-saving technology.
The Path Forward: Responsible development is crucial:
International Collaboration: Clear guidelines are needed for research and human trials.
Public Education: Open discussions ensure informed decisions about CRISPR.
Prioritize Safety and Ethics: Safety and ethical principles must be paramount.
CRISPR offers a powerful tool for a better future, but responsible development and addressing ethical concerns are essential. By prioritizing safety, fostering open dialogue, and ensuring equitable access, we can harness CRISPR's power for the benefit of all. (2998 characters)
The Importance of Community Nursing Care.pdfAD Healthcare
NDIS and Community 24/7 Nursing Care is a specific type of support that may be provided under the NDIS for individuals with complex medical needs who require ongoing nursing care in a community setting, such as their home or a supported accommodation facility.
CHAPTER 1 SEMESTER V PREVENTIVE-PEDIATRICS.pdfSachin Sharma
This content provides an overview of preventive pediatrics. It defines preventive pediatrics as preventing disease and promoting children's physical, mental, and social well-being to achieve positive health. It discusses antenatal, postnatal, and social preventive pediatrics. It also covers various child health programs like immunization, breastfeeding, ICDS, and the roles of organizations like WHO, UNICEF, and nurses in preventive pediatrics.
Explore our infographic on 'Essential Metrics for Palliative Care Management' which highlights key performance indicators crucial for enhancing the quality and efficiency of palliative care services.
This visual guide breaks down important metrics across four categories: Patient-Centered Metrics, Care Efficiency Metrics, Quality of Life Metrics, and Staff Metrics. Each section is designed to help healthcare professionals monitor and improve care delivery for patients facing serious illnesses. Understand how to implement these metrics in your palliative care practices for better outcomes and higher satisfaction levels.
CHAPTER 1 SEMESTER V - ROLE OF PEADIATRIC NURSE.pdfSachin Sharma
Pediatric nurses play a vital role in the health and well-being of children. Their responsibilities are wide-ranging, and their objectives can be categorized into several key areas:
1. Direct Patient Care:
Objective: Provide comprehensive and compassionate care to infants, children, and adolescents in various healthcare settings (hospitals, clinics, etc.).
This includes tasks like:
Monitoring vital signs and physical condition.
Administering medications and treatments.
Performing procedures as directed by doctors.
Assisting with daily living activities (bathing, feeding).
Providing emotional support and pain management.
2. Health Promotion and Education:
Objective: Promote healthy behaviors and educate children, families, and communities about preventive healthcare.
This includes tasks like:
Administering vaccinations.
Providing education on nutrition, hygiene, and development.
Offering breastfeeding and childbirth support.
Counseling families on safety and injury prevention.
3. Collaboration and Advocacy:
Objective: Collaborate effectively with doctors, social workers, therapists, and other healthcare professionals to ensure coordinated care for children.
Objective: Advocate for the rights and best interests of their patients, especially when children cannot speak for themselves.
This includes tasks like:
Communicating effectively with healthcare teams.
Identifying and addressing potential risks to child welfare.
Educating families about their child's condition and treatment options.
4. Professional Development and Research:
Objective: Stay up-to-date on the latest advancements in pediatric healthcare through continuing education and research.
Objective: Contribute to improving the quality of care for children by participating in research initiatives.
This includes tasks like:
Attending workshops and conferences on pediatric nursing.
Participating in clinical trials related to child health.
Implementing evidence-based practices into their daily routines.
By fulfilling these objectives, pediatric nurses play a crucial role in ensuring the optimal health and well-being of children throughout all stages of their development.
1. Plasmids
Indispensable tools that allow molecular biologists to obtain
essentially unlimited amounts of a DNA sequence
Small circular DNA molecules that replicate independently of the
host chromosomes
2. How are plasmids constructed?
What functional elements are found in our yeast
overexpression plasmids?
How are plasmids purified?
3. Plasmids used in molecular biology have been constructed in the lab
Molecular cloning
Enzymes are used to insert desired
pieces of foreign DNA into plasmids
Bacterial cells are transformed with
the plasmids. Copies of the
plasmids are purified from bacteria.
4. Shuttle vectors have origins of replication and selectable markers
for propagation in both bacteria and yeast
We are using plasmids that have been termed shuttle vectors,
because they can be propagated in either bacteria or yeast
Plasmids are propagated in
bacteria, which grow quickly
and maintain multiple copies
of the plasmids
non-pathogenic strain
of Escherichia coli
Saccharomyces cerevisiae
deletion strains
Plasmid-encoded genes
are expressed in yeast,
and phenotypes are
analyzed
5. How are plasmids constructed?
What functional elements are found in our yeast
overexpression plasmids?
How are plasmids purified?
6. 1 URA3
2 β-lactamase gene
3 pBR322 ori
4 yeast 2 µm origin
5 GAL1 promoter
6 C-terminal tags
1
2
3
4
5 6
ORF goes
here
*Plasmid names begin with a lower case “p”
The pBG1805*-derived plasmids are complex vectors designed
to express S. cerevisiae ORFs
7. pYES2.1-based plasmids used for S. pombe ORFs have many
elements in common with pBG1805-based plasmids
pYES2.1 (5886 bp)
used for S. pombe ORFs
1
2
3
4
5
7
2
1 3
4
5
6
URA3
β-lactamase gene
pBR322 ori
yeast 2 µm origin
GAL1 promoter
C-terminal tags
7
pBG1805 (6573 bp)
used for S. cerevisiae ORFs
1
2
3
4
5 6
ORF goes
here ORF goes
here
8. How are plasmids constructed?
What functional elements are found in our yeast
overexpression plasmids?
How are plasmids purified?
9. Plasmids are much smaller than bacterial
chromosomes
Plasmids are supercoiled in their native form
Supercoiling allows plasmids to renature quickly
after they are denatured
Plasmid purification is based on their distinctive
physical properties
Plasmids used in molecular biology are highly engineered and
contain elements of use to researchers
10. Plasmid purification from bacteria relies on their unique
physical properties
Bacterial cell with
plasmids
contains
MANY different,
well-folded proteins
1-2 copies of large
(>Mbp) , circular
bacterial DNA
complexed with
proteins
Multiple copies of small
(5-15 kbp) plasmids
Purification involves sequential denaturation and renaturation steps
11. Cells are first treated with base and a detergent
breaks open membrane
and denatures both DNA
and proteins
Proteins denature
irreversibly
Chromosomal DNA
denatures—will have
difficulty renaturing
because of its length
and many proteins
complexed to it
Plasmids denature, but
strands stay together
because of supercoiling
12. Extract is neutralized to allow DNA molecules to renature
Plasmids
renature and are
suspended in the
SUPERNATANT
following
centrifugation
Proteins and
chromosomal DNA form
aggregate irreversibly,
forming a PRECIPITATE
that can be collected by
centrifugation
When purifying plasmids, use a micropipette to remove the
supernatant for further processing steps
13. Zyppy purification kit use multiple steps to purify plasmids
Alkaline lysis
Neutralization
Purification of plasmid DNA on a silica resin
Elution of purified DNA from he silica resin
Let's look at the individual steps……………..
14. 1 Transformed E. coli cultures are
concentrated by centrifugation
2. The cell pellet is resuspended
in 600 µL TE buffer by vortexing
3. 100 µL of 7X Blue Zyppy lysis buffer
is added
0.1 N NaOH in buffer lyses the cells
GENTLY mix the contents by inverting the tube 4-6 times
Solution changes from cloudy to clear when cells are lysed
Warning: too much mechanical agitation can shear chromosomal DNA
Alkaline lysis
15. Neutralization
4. Add 350 µL yellow Zyppy
Neutralization buffer
Mix by inverting several times
A heavy precipitate will begin to form immediately!
The initial “glop” will become more granular when
neutralization is complete—but don’t overdo it!
The precipitate contains denatured proteins and
the denatured chromosomal DNA.
5. Spin down the denatured molecules
for 3 minutes at top speed. CAREFULLY
remove the supernatant containing the
plasmid – Don't be greedy! Purity is
preferred to yield!
16. Purification on
Zyppy silica resin
6. Apply the supernatant to the spin
column. Place the column in the
collection tube. Centrifuge the column
for ~15 seconds at top speed.
7. Discard the flow through in the
collection tube. Add 200 µL Zyppy
EndoWash. Centrifuge ~15 sec.
EndoWash contains guanidine
hydrochloride and
isopropanol. It removes
contaminating proteins that
are bound to the resin.
8. Discard the flow through in the
collection tube. Add 400 µL Column
Wash. Centrifuge 1 min.
17. Plasmid Elution
9. Transfer the column to a clean,
LABELED microcentrifuge tube
10. Add 50 µL TE buffer directly on
top of the column. Allow the column to
stand upright in the test tube for ~10
min. (Plasmid is being eluted.)
11. Spin the column for 30 seconds.
Plasmid DNA will be collected in the
microcentrifuge tube. Pure plasmid DNA
collects here!