The document summarizes a summer training presentation on understanding the life cycle of Dictyostelium discoideum and cloning of a putative gene. Key steps included optimizing PCR to amplify the gene segment, digesting the PCR product and vector with restriction enzymes, ligating the insert into the vector, transforming bacteria, and isolating the recombinant plasmid to check for proper ligation of the insert. The goal was to clone two segments of the putative gene into a pDrive vector to generate a knockout.
Communication is the key factor in the success of any organization. When it comes to effective communication, there are certain barriers that every organization faces. People often feel that communication is as easy and simple as it sounds. No doubt, but what makes it complex, difficult and frustrating are the barriers that come in its way. Here are a few do's and don'ts to remove or reduce these barriers.
2024 State of Marketing Report – by HubspotMarius Sescu
https://www.hubspot.com/state-of-marketing
· Scaling relationships and proving ROI
· Social media is the place for search, sales, and service
· Authentic influencer partnerships fuel brand growth
· The strongest connections happen via call, click, chat, and camera.
· Time saved with AI leads to more creative work
· Seeking: A single source of truth
· TLDR; Get on social, try AI, and align your systems.
· More human marketing, powered by robots
ChatGPT is a revolutionary addition to the world since its introduction in 2022. A big shift in the sector of information gathering and processing happened because of this chatbot. What is the story of ChatGPT? How is the bot responding to prompts and generating contents? Swipe through these slides prepared by Expeed Software, a web development company regarding the development and technical intricacies of ChatGPT!
Product Design Trends in 2024 | Teenage EngineeringsPixeldarts
The realm of product design is a constantly changing environment where technology and style intersect. Every year introduces fresh challenges and exciting trends that mold the future of this captivating art form. In this piece, we delve into the significant trends set to influence the look and functionality of product design in the year 2024.
Communication is the key factor in the success of any organization. When it comes to effective communication, there are certain barriers that every organization faces. People often feel that communication is as easy and simple as it sounds. No doubt, but what makes it complex, difficult and frustrating are the barriers that come in its way. Here are a few do's and don'ts to remove or reduce these barriers.
2024 State of Marketing Report – by HubspotMarius Sescu
https://www.hubspot.com/state-of-marketing
· Scaling relationships and proving ROI
· Social media is the place for search, sales, and service
· Authentic influencer partnerships fuel brand growth
· The strongest connections happen via call, click, chat, and camera.
· Time saved with AI leads to more creative work
· Seeking: A single source of truth
· TLDR; Get on social, try AI, and align your systems.
· More human marketing, powered by robots
ChatGPT is a revolutionary addition to the world since its introduction in 2022. A big shift in the sector of information gathering and processing happened because of this chatbot. What is the story of ChatGPT? How is the bot responding to prompts and generating contents? Swipe through these slides prepared by Expeed Software, a web development company regarding the development and technical intricacies of ChatGPT!
Product Design Trends in 2024 | Teenage EngineeringsPixeldarts
The realm of product design is a constantly changing environment where technology and style intersect. Every year introduces fresh challenges and exciting trends that mold the future of this captivating art form. In this piece, we delve into the significant trends set to influence the look and functionality of product design in the year 2024.
How Race, Age and Gender Shape Attitudes Towards Mental HealthThinkNow
Mental health has been in the news quite a bit lately. Dozens of U.S. states are currently suing Meta for contributing to the youth mental health crisis by inserting addictive features into their products, while the U.S. Surgeon General is touring the nation to bring awareness to the growing epidemic of loneliness and isolation. The country has endured periods of low national morale, such as in the 1970s when high inflation and the energy crisis worsened public sentiment following the Vietnam War. The current mood, however, feels different. Gallup recently reported that national mental health is at an all-time low, with few bright spots to lift spirits.
To better understand how Americans are feeling and their attitudes towards mental health in general, ThinkNow conducted a nationally representative quantitative survey of 1,500 respondents and found some interesting differences among ethnic, age and gender groups.
Technology
For example, 52% agree that technology and social media have a negative impact on mental health, but when broken out by race, 61% of Whites felt technology had a negative effect, and only 48% of Hispanics thought it did.
While technology has helped us keep in touch with friends and family in faraway places, it appears to have degraded our ability to connect in person. Staying connected online is a double-edged sword since the same news feed that brings us pictures of the grandkids and fluffy kittens also feeds us news about the wars in Israel and Ukraine, the dysfunction in Washington, the latest mass shooting and the climate crisis.
Hispanics may have a built-in defense against the isolation technology breeds, owing to their large, multigenerational households, strong social support systems, and tendency to use social media to stay connected with relatives abroad.
Age and Gender
When asked how individuals rate their mental health, men rate it higher than women by 11 percentage points, and Baby Boomers rank it highest at 83%, saying it’s good or excellent vs. 57% of Gen Z saying the same.
Gen Z spends the most amount of time on social media, so the notion that social media negatively affects mental health appears to be correlated. Unfortunately, Gen Z is also the generation that’s least comfortable discussing mental health concerns with healthcare professionals. Only 40% of them state they’re comfortable discussing their issues with a professional compared to 60% of Millennials and 65% of Boomers.
Race Affects Attitudes
As seen in previous research conducted by ThinkNow, Asian Americans lag other groups when it comes to awareness of mental health issues. Twenty-four percent of Asian Americans believe that having a mental health issue is a sign of weakness compared to the 16% average for all groups. Asians are also considerably less likely to be aware of mental health services in their communities (42% vs. 55%) and most likely to seek out information on social media (51% vs. 35%).
AI Trends in Creative Operations 2024 by Artwork Flow.pdfmarketingartwork
This article is all about what AI trends will emerge in the field of creative operations in 2024. All the marketers and brand builders should be aware of these trends for their further use and save themselves some time!
A report by thenetworkone and Kurio.
The contributing experts and agencies are (in an alphabetical order): Sylwia Rytel, Social Media Supervisor, 180heartbeats + JUNG v MATT (PL), Sharlene Jenner, Vice President - Director of Engagement Strategy, Abelson Taylor (USA), Alex Casanovas, Digital Director, Atrevia (ES), Dora Beilin, Senior Social Strategist, Barrett Hoffher (USA), Min Seo, Campaign Director, Brand New Agency (KR), Deshé M. Gully, Associate Strategist, Day One Agency (USA), Francesca Trevisan, Strategist, Different (IT), Trevor Crossman, CX and Digital Transformation Director; Olivia Hussey, Strategic Planner; Simi Srinarula, Social Media Manager, The Hallway (AUS), James Hebbert, Managing Director, Hylink (CN / UK), Mundy Álvarez, Planning Director; Pedro Rojas, Social Media Manager; Pancho González, CCO, Inbrax (CH), Oana Oprea, Head of Digital Planning, Jam Session Agency (RO), Amy Bottrill, Social Account Director, Launch (UK), Gaby Arriaga, Founder, Leonardo1452 (MX), Shantesh S Row, Creative Director, Liwa (UAE), Rajesh Mehta, Chief Strategy Officer; Dhruv Gaur, Digital Planning Lead; Leonie Mergulhao, Account Supervisor - Social Media & PR, Medulla (IN), Aurelija Plioplytė, Head of Digital & Social, Not Perfect (LI), Daiana Khaidargaliyeva, Account Manager, Osaka Labs (UK / USA), Stefanie Söhnchen, Vice President Digital, PIABO Communications (DE), Elisabeth Winiartati, Managing Consultant, Head of Global Integrated Communications; Lydia Aprina, Account Manager, Integrated Marketing and Communications; Nita Prabowo, Account Manager, Integrated Marketing and Communications; Okhi, Web Developer, PNTR Group (ID), Kei Obusan, Insights Director; Daffi Ranandi, Insights Manager, Radarr (SG), Gautam Reghunath, Co-founder & CEO, Talented (IN), Donagh Humphreys, Head of Social and Digital Innovation, THINKHOUSE (IRE), Sarah Yim, Strategy Director, Zulu Alpha Kilo (CA).
Trends In Paid Search: Navigating The Digital Landscape In 2024Search Engine Journal
The search marketing landscape is evolving rapidly with new technologies, and professionals, like you, rely on innovative paid search strategies to meet changing demands.
It’s important that you’re ready to implement new strategies in 2024.
Check this out and learn the top trends in paid search advertising that are expected to gain traction, so you can drive higher ROI more efficiently in 2024.
You’ll learn:
- The latest trends in AI and automation, and what this means for an evolving paid search ecosystem.
- New developments in privacy and data regulation.
- Emerging ad formats that are expected to make an impact next year.
Watch Sreekant Lanka from iQuanti and Irina Klein from OneMain Financial as they dive into the future of paid search and explore the trends, strategies, and technologies that will shape the search marketing landscape.
If you’re looking to assess your paid search strategy and design an industry-aligned plan for 2024, then this webinar is for you.
5 Public speaking tips from TED - Visualized summarySpeakerHub
From their humble beginnings in 1984, TED has grown into the world’s most powerful amplifier for speakers and thought-leaders to share their ideas. They have over 2,400 filmed talks (not including the 30,000+ TEDx videos) freely available online, and have hosted over 17,500 events around the world.
With over one billion views in a year, it’s no wonder that so many speakers are looking to TED for ideas on how to share their message more effectively.
The article “5 Public-Speaking Tips TED Gives Its Speakers”, by Carmine Gallo for Forbes, gives speakers five practical ways to connect with their audience, and effectively share their ideas on stage.
Whether you are gearing up to get on a TED stage yourself, or just want to master the skills that so many of their speakers possess, these tips and quotes from Chris Anderson, the TED Talks Curator, will encourage you to make the most impactful impression on your audience.
See the full article and more summaries like this on SpeakerHub here: https://speakerhub.com/blog/5-presentation-tips-ted-gives-its-speakers
See the original article on Forbes here:
http://www.forbes.com/forbes/welcome/?toURL=http://www.forbes.com/sites/carminegallo/2016/05/06/5-public-speaking-tips-ted-gives-its-speakers/&refURL=&referrer=#5c07a8221d9b
ChatGPT and the Future of Work - Clark Boyd Clark Boyd
Everyone is in agreement that ChatGPT (and other generative AI tools) will shape the future of work. Yet there is little consensus on exactly how, when, and to what extent this technology will change our world.
Businesses that extract maximum value from ChatGPT will use it as a collaborative tool for everything from brainstorming to technical maintenance.
For individuals, now is the time to pinpoint the skills the future professional will need to thrive in the AI age.
Check out this presentation to understand what ChatGPT is, how it will shape the future of work, and how you can prepare to take advantage.
A brief introduction to DataScience with explaining of the concepts, algorithms, machine learning, supervised and unsupervised learning, clustering, statistics, data preprocessing, real-world applications etc.
It's part of a Data Science Corner Campaign where I will be discussing the fundamentals of DataScience, AIML, Statistics etc.
Time Management & Productivity - Best PracticesVit Horky
Here's my presentation on by proven best practices how to manage your work time effectively and how to improve your productivity. It includes practical tips and how to use tools such as Slack, Google Apps, Hubspot, Google Calendar, Gmail and others.
The six step guide to practical project managementMindGenius
The six step guide to practical project management
If you think managing projects is too difficult, think again.
We’ve stripped back project management processes to the
basics – to make it quicker and easier, without sacrificing
the vital ingredients for success.
“If you’re looking for some real-world guidance, then The Six Step Guide to Practical Project Management will help.”
Dr Andrew Makar, Tactical Project Management
Unlocking the Power of ChatGPT and AI in Testing - A Real-World Look, present...Applitools
During this webinar, Anand Bagmar demonstrates how AI tools such as ChatGPT can be applied to various stages of the software development life cycle (SDLC) using an eCommerce application case study. Find the on-demand recording and more info at https://applitools.info/b59
Key takeaways:
• Learn how to use ChatGPT to add AI power to your testing and test automation
• Understand the limitations of the technology and where human expertise is crucial
• Gain insight into different AI-based tools
• Adopt AI-based tools to stay relevant and optimize work for developers and testers
* ChatGPT and OpenAI belong to OpenAI, L.L.C.
How Race, Age and Gender Shape Attitudes Towards Mental HealthThinkNow
Mental health has been in the news quite a bit lately. Dozens of U.S. states are currently suing Meta for contributing to the youth mental health crisis by inserting addictive features into their products, while the U.S. Surgeon General is touring the nation to bring awareness to the growing epidemic of loneliness and isolation. The country has endured periods of low national morale, such as in the 1970s when high inflation and the energy crisis worsened public sentiment following the Vietnam War. The current mood, however, feels different. Gallup recently reported that national mental health is at an all-time low, with few bright spots to lift spirits.
To better understand how Americans are feeling and their attitudes towards mental health in general, ThinkNow conducted a nationally representative quantitative survey of 1,500 respondents and found some interesting differences among ethnic, age and gender groups.
Technology
For example, 52% agree that technology and social media have a negative impact on mental health, but when broken out by race, 61% of Whites felt technology had a negative effect, and only 48% of Hispanics thought it did.
While technology has helped us keep in touch with friends and family in faraway places, it appears to have degraded our ability to connect in person. Staying connected online is a double-edged sword since the same news feed that brings us pictures of the grandkids and fluffy kittens also feeds us news about the wars in Israel and Ukraine, the dysfunction in Washington, the latest mass shooting and the climate crisis.
Hispanics may have a built-in defense against the isolation technology breeds, owing to their large, multigenerational households, strong social support systems, and tendency to use social media to stay connected with relatives abroad.
Age and Gender
When asked how individuals rate their mental health, men rate it higher than women by 11 percentage points, and Baby Boomers rank it highest at 83%, saying it’s good or excellent vs. 57% of Gen Z saying the same.
Gen Z spends the most amount of time on social media, so the notion that social media negatively affects mental health appears to be correlated. Unfortunately, Gen Z is also the generation that’s least comfortable discussing mental health concerns with healthcare professionals. Only 40% of them state they’re comfortable discussing their issues with a professional compared to 60% of Millennials and 65% of Boomers.
Race Affects Attitudes
As seen in previous research conducted by ThinkNow, Asian Americans lag other groups when it comes to awareness of mental health issues. Twenty-four percent of Asian Americans believe that having a mental health issue is a sign of weakness compared to the 16% average for all groups. Asians are also considerably less likely to be aware of mental health services in their communities (42% vs. 55%) and most likely to seek out information on social media (51% vs. 35%).
AI Trends in Creative Operations 2024 by Artwork Flow.pdfmarketingartwork
This article is all about what AI trends will emerge in the field of creative operations in 2024. All the marketers and brand builders should be aware of these trends for their further use and save themselves some time!
A report by thenetworkone and Kurio.
The contributing experts and agencies are (in an alphabetical order): Sylwia Rytel, Social Media Supervisor, 180heartbeats + JUNG v MATT (PL), Sharlene Jenner, Vice President - Director of Engagement Strategy, Abelson Taylor (USA), Alex Casanovas, Digital Director, Atrevia (ES), Dora Beilin, Senior Social Strategist, Barrett Hoffher (USA), Min Seo, Campaign Director, Brand New Agency (KR), Deshé M. Gully, Associate Strategist, Day One Agency (USA), Francesca Trevisan, Strategist, Different (IT), Trevor Crossman, CX and Digital Transformation Director; Olivia Hussey, Strategic Planner; Simi Srinarula, Social Media Manager, The Hallway (AUS), James Hebbert, Managing Director, Hylink (CN / UK), Mundy Álvarez, Planning Director; Pedro Rojas, Social Media Manager; Pancho González, CCO, Inbrax (CH), Oana Oprea, Head of Digital Planning, Jam Session Agency (RO), Amy Bottrill, Social Account Director, Launch (UK), Gaby Arriaga, Founder, Leonardo1452 (MX), Shantesh S Row, Creative Director, Liwa (UAE), Rajesh Mehta, Chief Strategy Officer; Dhruv Gaur, Digital Planning Lead; Leonie Mergulhao, Account Supervisor - Social Media & PR, Medulla (IN), Aurelija Plioplytė, Head of Digital & Social, Not Perfect (LI), Daiana Khaidargaliyeva, Account Manager, Osaka Labs (UK / USA), Stefanie Söhnchen, Vice President Digital, PIABO Communications (DE), Elisabeth Winiartati, Managing Consultant, Head of Global Integrated Communications; Lydia Aprina, Account Manager, Integrated Marketing and Communications; Nita Prabowo, Account Manager, Integrated Marketing and Communications; Okhi, Web Developer, PNTR Group (ID), Kei Obusan, Insights Director; Daffi Ranandi, Insights Manager, Radarr (SG), Gautam Reghunath, Co-founder & CEO, Talented (IN), Donagh Humphreys, Head of Social and Digital Innovation, THINKHOUSE (IRE), Sarah Yim, Strategy Director, Zulu Alpha Kilo (CA).
Trends In Paid Search: Navigating The Digital Landscape In 2024Search Engine Journal
The search marketing landscape is evolving rapidly with new technologies, and professionals, like you, rely on innovative paid search strategies to meet changing demands.
It’s important that you’re ready to implement new strategies in 2024.
Check this out and learn the top trends in paid search advertising that are expected to gain traction, so you can drive higher ROI more efficiently in 2024.
You’ll learn:
- The latest trends in AI and automation, and what this means for an evolving paid search ecosystem.
- New developments in privacy and data regulation.
- Emerging ad formats that are expected to make an impact next year.
Watch Sreekant Lanka from iQuanti and Irina Klein from OneMain Financial as they dive into the future of paid search and explore the trends, strategies, and technologies that will shape the search marketing landscape.
If you’re looking to assess your paid search strategy and design an industry-aligned plan for 2024, then this webinar is for you.
5 Public speaking tips from TED - Visualized summarySpeakerHub
From their humble beginnings in 1984, TED has grown into the world’s most powerful amplifier for speakers and thought-leaders to share their ideas. They have over 2,400 filmed talks (not including the 30,000+ TEDx videos) freely available online, and have hosted over 17,500 events around the world.
With over one billion views in a year, it’s no wonder that so many speakers are looking to TED for ideas on how to share their message more effectively.
The article “5 Public-Speaking Tips TED Gives Its Speakers”, by Carmine Gallo for Forbes, gives speakers five practical ways to connect with their audience, and effectively share their ideas on stage.
Whether you are gearing up to get on a TED stage yourself, or just want to master the skills that so many of their speakers possess, these tips and quotes from Chris Anderson, the TED Talks Curator, will encourage you to make the most impactful impression on your audience.
See the full article and more summaries like this on SpeakerHub here: https://speakerhub.com/blog/5-presentation-tips-ted-gives-its-speakers
See the original article on Forbes here:
http://www.forbes.com/forbes/welcome/?toURL=http://www.forbes.com/sites/carminegallo/2016/05/06/5-public-speaking-tips-ted-gives-its-speakers/&refURL=&referrer=#5c07a8221d9b
ChatGPT and the Future of Work - Clark Boyd Clark Boyd
Everyone is in agreement that ChatGPT (and other generative AI tools) will shape the future of work. Yet there is little consensus on exactly how, when, and to what extent this technology will change our world.
Businesses that extract maximum value from ChatGPT will use it as a collaborative tool for everything from brainstorming to technical maintenance.
For individuals, now is the time to pinpoint the skills the future professional will need to thrive in the AI age.
Check out this presentation to understand what ChatGPT is, how it will shape the future of work, and how you can prepare to take advantage.
A brief introduction to DataScience with explaining of the concepts, algorithms, machine learning, supervised and unsupervised learning, clustering, statistics, data preprocessing, real-world applications etc.
It's part of a Data Science Corner Campaign where I will be discussing the fundamentals of DataScience, AIML, Statistics etc.
Time Management & Productivity - Best PracticesVit Horky
Here's my presentation on by proven best practices how to manage your work time effectively and how to improve your productivity. It includes practical tips and how to use tools such as Slack, Google Apps, Hubspot, Google Calendar, Gmail and others.
The six step guide to practical project managementMindGenius
The six step guide to practical project management
If you think managing projects is too difficult, think again.
We’ve stripped back project management processes to the
basics – to make it quicker and easier, without sacrificing
the vital ingredients for success.
“If you’re looking for some real-world guidance, then The Six Step Guide to Practical Project Management will help.”
Dr Andrew Makar, Tactical Project Management
Unlocking the Power of ChatGPT and AI in Testing - A Real-World Look, present...Applitools
During this webinar, Anand Bagmar demonstrates how AI tools such as ChatGPT can be applied to various stages of the software development life cycle (SDLC) using an eCommerce application case study. Find the on-demand recording and more info at https://applitools.info/b59
Key takeaways:
• Learn how to use ChatGPT to add AI power to your testing and test automation
• Understand the limitations of the technology and where human expertise is crucial
• Gain insight into different AI-based tools
• Adopt AI-based tools to stay relevant and optimize work for developers and testers
* ChatGPT and OpenAI belong to OpenAI, L.L.C.
2. Understanding of the life cycle of Dictyostelium
discoideum and cloning of putative gene
Mentor:
Dr. Shweta Saran
Developmental Biology Lab
SLS JNU, New Delhi.
Presented By:
Bhupender Verma
M.Sc. (F) MHG BHU
3. Introduction
Dictyostelium discoideum are single
cellular organisms
Live on decaying logs and feed on
bacteria
Dictyostelium discoideum follow asexual
cycle-
• when there is plenty of food available
• Live solitary
• Are haploid
• Reproduce by binary fission
Also called as “Social amoebae”
During starvation organisms aggregate
and migrating slug is formed that
culminates in a fruiting body to release
spores
6. Life cycle
In lab Dicty are grown in HL5 liquid media at 22ºC.
In nutrient rich conditions Dicty divide by binary fission
and remain solitary.
For studying their development a concentrated solution
is transferred to the NNA plates.
In the absence of nutrients, these myxomoebae join
together to form moving streams of cells converging at a
central point.
Different developmental stages that can be observed:
• Tight aggregate
• Migrating slug (pseudoplasmodium or grex) – slimy
sheath encased
• Fruiting body
7. Vegetative loose aggregate mound migratory slug
Early culminant fruiting body
8. Cloning of the putative gene:
Strategy:
• Design the primers
• Gradient PCR for optimizing annealing conditions
• Standard PCR for obtaining amplified insert
• Digestion of vector and insert with HindIII and PstI
• Liagtion of insert to the vector
• Transform bacteria with recombinant vector
• Isolate cloned plasmid and digest for checking if insert is
desirable
9. Gradient PCR for optimizing annealing conditions:
1 2 3 4 5 6 7 8 9 10 M 11 12 13 14 15 16 17 18 19 20 Design the primers
• Gradient PCR
5 kb • Standard PCR
1.5 kb • Digestion with HindIII
500 bp and PstI
• Liagtion
• Bacterial
21 22 23 24 25 26 27 28 29 30 M 31 32 33 34 35 36
transformaton
• Isolate cloned plasmid
5 kb
and digest for checking
1.5 kb if insert is desirable
500 bp
10. Standard PCR for obtaining amplified insert:
Design the primers
• Gradient PCR
• Standard PCR
• Digestion with HindIII
5 kb and PstI
• Liagtion
1.5 kb • Bacterial
transformaton
500 bp • Isolate cloned plasmid
and digest for checking
if insert is desirable
11. pDrive cloning vector-
Design the primers
• Gradient PCR
• Standard PCR
• Digestion with HindIII
and PstI
• Liagtion
• Bacterial
transformaton
• Isolate cloned plasmid
and digest for checking
if insert is desirable
12. Digestion of vector and insert with HindIII and PstI
Design the primers
• Gradient PCR
• Standard PCR
• Digestion with HindIII
and PstI
5 kb • Liagtion
• Bacterial
1.5 kb transformaton
• Isolate cloned plasmid
and digest for checking
500 bp
if insert is desirable
13. Checking insert from fallout size:
Design the primers
5 kb • Gradient PCR
1.5 kb • Standard PCR
500 bp • Digestion with HindIII
and PstI
• Liagtion
• Bacterial
transformaton
5 kb
• Isolate cloned plasmid
1.5 kb
and digest for checking
500 bp if insert is desirable
14. Cloning two segments of a putative gene
in pDrive vector for making Knockout-
1. Insert 1 – obtained from digestion (HindIII & NotI) of cloning
vector as pop out
2. pDrive vector – already had other insert so digest with (HindIII &
NotI) & obtain vector-BSR
3. Ligate insert to vector
4. Transform bacteria
5. Grow bacteria and isolate plasmid
6. Digest plasmid for checking proper ligation
7. Proceed with the colony that had proper insert
8. PCR of insert 2
9. Digest insert 2
10.Ligation
11.Transformation
12.Isolate plasmid and check
15. 1. Insert 1-as pop out
Digestion with HindIII & NotI 2. pDrive -had other
insert
3. Ligation of insert 1
4. Transform E.coli
5. Grow E.coli &
isolate plasmid
6. Digest plasmid for
check
7. Proceed with sample
+BSR having proper insert
8. PCR of insert 2
9. Digest insert 2
10.Ligation
11.Transformation
12.Isolate plasmid and
check
16. 1. Insert 1-as pop out
Check after elution from 2. pDrive -had other
agarose gel: insert
3. Ligation of insert 1
4. Transform E.coli
5. Grow E.coli &
isolate plasmid
6. Digest plasmid for
5 kb
check
7. Proceed with sample
1.5 kb having proper insert
8. PCR of insert 2
500 bp 9. Digest insert 2
10.Ligation
11.Transformation
12.Isolate plasmid and
check
17. 1. Insert 1-as pop out
For checking again digest 2. pDrive -had other
with HindII & NotI insert
3. Ligation of insert 1
4. Transform E.coli
5 kb 5. Grow E.coli &
1.5 kb isolate plasmid
500 bp 6. Digest plasmid for
check
7. Proceed with sample
having proper insert
8. PCR of insert 2
5 kb 9. Digest insert 2
1.5 kb 10.Ligation
500 bp 11.Transformation
12.Isolate plasmid and
check
18. 1. Insert 1-as pop out
For checking again digest 2. pDrive -had other
with HindII & NotI insert
3. Ligation of insert 1
4. Transform E.coli
5 kb 5. Grow E.coli &
1.5 kb isolate plasmid
500 bp 6. Digest plasmid for
check
7. Proceed with sample
having proper insert
8. PCR of insert 2
5 kb 9. Digest insert 2
1.5 kb 10.Ligation
500 bp 11.Transformation
12.Isolate plasmid and
check
20. 1. Insert 1-as pop out
Digestion with HindIII & 2. pDrive -had other
BamhI: insert
3. Ligation of insert 1
4. Transform E.coli
5. Grow E.coli &
isolate plasmid
6. Digest plasmid for
check
7. Proceed with sample
5 kb having proper insert
1.5 kb 8. PCR of insert 2
500 bp 9. Digestion of insert 2
10.Ligation
11.Transformation
12.Isolate plasmid and
check
21. 1. Insert 1-as pop out
Result: 2. pDrive -had other
insert
3. Ligation of insert 1
4. Transform E.coli
5. Grow E.coli &
pDrive Insert1-BSR-Insert2
isolate plasmid
6. Digest plasmid for
check
7. Proceed with sample
Now we are ready with a DNA having proper insert
8. PCR of insert 2
segment that is “insert1-BSR-
9. Digestion of insert 2
insert2”
10.Ligation
This insert can be used for 11.Transformation
knocking out this putative genes 12.Isolate plasmid and
And my insert is still under study check
22. References:
RICARDO ESCALANTE and JUAN J. VICENTE- Dictyostelium
discoideum: a model system for differentiation and patterning(2000)
L. Eichinger, J. A. Pachebat, G. Glockner- The genome of the social
amoeba Dictyostelium discoideum (2005)
Developmental biology- Scott F. Gilbert- 8th edition.