Adieu.

 And she went her way

The way, she was meant to go.

Laid peacefully among the autumn floor

Rain thudding over her coffin's door

To a place where the living cannot be

miles away where I cannot see

Oh what peace has death bought upon thee

Freed of malice and despair you flee

As much as I wish you'll come back again

I know you're far from me seeing you again

I allow myself a little pain

Though I know it's all in vain

I wish I said a parting goodbye

But in hope, I look up into the bluest sky. 

I miss you.

© Vidya 5.6.21

Cheers to the end and to a new beginning...!

This year-end is a special one. We are bound to be more thankful than ever before. Each of us. And yes! I am.

For me, 2020 began with a promise card that said, "I will be with thee" - Genesis 28:15. The promise has been kept and fulfilled. Not for a moment has God left my side the whole year. Every prayer has been answered, every harm kept away, every cry has been heard. The Lord is truly faithful and a promise keeper. As I rewind everything that has happened in the year, I can only thank The Lord. He has kept His promise. While the world turned upside down every way, He's given shelter under his mighty wings and I can't be enough thankful for His never-ending grace, mercy, kindness and Goodness.

Some of us have lost our loved ones, some lost sources of livelihood, some have sunk into depression, faced economic lows, emotional breakdowns and everything negative that could've been thought of. But dearest, among everything odd, we've got to admit the fact that the year 2020 has taught us and allowed us to count our blessings. We've survived this, We've helped, we've been helped, while we've seen and heard which was "never before" and hope it'll be a "never after". 

Here's a mandatory long Thank You note to all the doctors, medical staff and workers who've fought the fight with an "unheard of" spirit and effort. Gratitude to everyone who's taken extraordinary measures to offer help to the needful. Professors and teachers who've done an extremely commendable job to keep the teaching-learning in the best possible way. Time to remember our families, who've stayed with us in the unimaginable, unrealistically real times. Relatives who've looked out for each other and prayed for our families. Thanks to friends, who've kept in touch and did not mind sending how-about messages no matter how forgetful I've been. To everyone who has spread positivity, happy vibes and warmth, A heartfelt thank you. Because every little thing did matter.

Don't forget to give yourself a pat. In the midst of all the storm and the tumult, you've been strong. You've kept the smile on. Made someone or the other smile. You've made it to the very end of 2020 and not given up. That's an achievement. Take a bow. 

Happy New Year 2021

It doesn't take much

This morning, I woke up to a calendar notification that read, "World Kindness Day" that made me think when was the last time I was kind to someone. My brain wouldn't be quiet. An here I am, writing down to ease the imaginary pressure on what's enclosed in my cranium. 

Being kind is so simple yet so strong. Acts of kindness hack the norm- "everything fades with time". They just remain, even more, when you are on the receiving end. During primary school, I would go to school on public transport. There would be just one bus in the morning that would drop me right in front of my school. To catch the bus, it was a 7-8 minute walk to the bus stop. This one driver who used to drive the bus 5 out of six weekdays would be so kind to stop the bus right in front of my house even in heavy traffic. That's the simplest act of kindness he would show to me being in that profession. We would just smile at each other and that's it. A good day to start. He might never even remember that he's done it but I would cherish it for a long long time. Our canteen Uncle, who would give a free choco- stick on birthdays,  the butcher who would give leftovers to street dogs, an old lady at the corner of the street who would ask how-abouts of passers-by are all remembered for the small things they did. 

We all talk about changing the world in so many ways, it's time we realize it just takes simple, inexpensive, little efforts to make a lot of difference. Being kind is instinctive, intuitive, it doesn't need a lot of planning or effort. It's just caring for others. Smile. Appreciate. Give. Thank. All unconditionally. Our simple gestures may make someone's day a little brighter, a lot happier. All of us realize how hard our responsibilities are to carry. We race with time and there's no excuse. But, it doesn't take a lot of time to say a few good words to the person who cooked for you today, send a quick text to an old friend while riding an elevator, hold an oldie's hand at the crossroad, smile to a familiar face on your street and the list goes on. There are no rules to follow, no books to learn from, no boundaries to be bound by for being kind. Let's use our profession to be kind to so many people in so many ways. Unknowingly, you might be the reason for someone not giving up today, for deciding to push a little more, to pass on kindness to someone else. 

After all, one day, life has to stop and it would be lovely to be remembered for how kind we were rather than how busy we spent. 

Just Be Kind. 

The Untold Origin of Face Masks

Negate the negativity

Solving math problem in childhood, I would always wonder and get confused why in a mathematical operation, a negative sign would change everything to negative! I would never get it right. Why was this negative sign always making my positive numbers negative and reducing their value? Okay, if that was a little confusing, here's a situation:  4 + 6 = 10 ; 4 + (- 6) =  -2. Why? Why did the negativity carried by 6 had to reduce the value of four? Alright. No more math. The point I wanted to make was, our negative thoughts, negative vibes and negative impressions we carry become a burden altogether. The negatives are so powerful they'd reduce your positivity to zero or even less. Life isn't so worthless to let it be shadowed by negative thoughts. Don't you think? And it is not that difficult to overpower the devil. Why? doesn't a positive terminal always accompany a negative terminal? It's just that you need to flip it around and look at it in a different view. Here's some math again, 4 - (-6) =10; Try negating the negativity that 6 carries. Isn't it simple? It may not be as simple as the math in real-life situations, but nevertheless, it isn't too hard either. Traceback the root cause for your negativity, a person? your goal? your failures? your career? Hey there, you are strong, bold and courageous enough to break the walls you've yourself created by placing those negatives around you. Smash them all. Get free. It's not ok to remain negative, to carry the guilt, to doubt yourself, to stop where you are. Just a step away and bang! Light will enter. Say Good-bye to whatever you've been holding on to that was hurting. If it's a failure, make it a stepping stone to your success, if it's something you're guilty about, there's nothing to be done, move ahead, if it's your past, learn your lesson and head straight. No matter what grab the next chance life's offering. And MARCH. No stopping and turning back. 

Everything at a standstill.

Down the lane,
We would be narrating our lockdown days to our children, to our newer generations. Telling them how the tiniest virus got us all caged. Even with plentiful of everything, the greatest of the nations stood vulnerable to the virus, helpless, seeking help, wanting to help, yet everyone locked up to themselves. A little more than a month ago, the world was still normal. Booming with business, the universities running around with their busy schedules, technology freaks breaking their heads over codes, people planning their journeys abroad, daily wage workers struggling hard, thieves stealing, everything was just as it was supposed to be. And then the virus sneaked in.

Just like the air that has no boundaries, no direction, omnipresent, the virus went to and fro, and a wave of COVID-19 cases drenched almost all the countries. In the generation of globalization, the country's borders shut close, the ever-busy roads now with deafening silence and the ever-booming offices at standstill, never imaginable consequences! With the death song that arose in the East, all the other nations froze. It became convinceable that the virus had no mercy, it hit the rich, it swept the poor, it entered the villas, it ruled the slums. While the 21st-century man thought he was unstoppable, best than never before, a generation that could move any boulder, reaching the unreachable, achieving the impossible and doing the unimaginable, now its all a standstill. The medicines won't work. no treatment to the ailment, it's just a natural recovery that could get you better. No political party to blame for the issue, no one to be accused, no culprit for the natural massacre. But just watch it stealthily spread across the borders.
Not far from today, we would be teaching children in schools that wars are not always fought on the battlefield with swords, firing guns or explosives, neither are the wars against countries or within the nations. There are wars, like the one the man-kind is fighting today with doctors on the front-line, cops right behind to make sure everything goes well, the governments making strategies to fight with the unseen and every individual committed to locking themselves in their places with only one slogan everywhere, "Stay home, Stay safe" was all part of the new strategy to win the war.
It's a lot to take in for everyone at the moment that we aren't well equipped for the unforeseen situation that has arisen out of nowhere in a time-period where research and medical field around the world were at their best uncovering what was indecipherable. Humanity fights to come back.
With prayers and faith in God rising, trusting in the help that's already on its way, we hope, "This too shall pass".

Who would guard the guardians- A review of Digital Fortress by Dan Brown

It all starts with a University professor, David Becker's phone call to his fiance, Susan Fletcher, saying there's a change in their plans and it wouldn't be possible to have the sweet picnic as he has to fly to Spain to attend for an emergency. 

The plot is set up in  U.S's National Security Agency(NSA) which is a top-secret organization which has a program that can track personal conversations sent through Emails. They take pride in owning a computer called TRANSLTR which is programmed to unscramble any code but its presence is masked from the public as there were outcries regarding the privacy of common citizens. It helped the NSA in saving the nation from terrorist attacks and several safety issues. Susan is the head of the cryptography section at NSA who is carefully brought up by the director, Strathmore by protecting her in the workplace and helping her grow careerwise ( He gave me an impression of an ideal employer but!! ). 

The Agency has encountered a code that's been running on TRANSLTR since 15 hours, yet remains undecrypted. This is seen as no small issue as it questions the efficiency of TRANSLTR and on a serious note, Susan is called to the office on a Saturday morning by Strathmore slightly after her call with David which has already left her in a bad mood. 

Dan Brown takes us through a thriller journey where quite a handful of murders are encountered, half of the book has its plot in Spain whereas the other half is plotted at the NSA head office. Unknowingly, both David and Susan are on the same mission employed by the same person, Strathmore but planned to have different fates. David is sent to be killed while Susan is being used to serve some other purpose. The rollercoaster ride through the book leaves the reader with nail-biting suspense, exciting real-time digital emergency and a number of what-ifs. 

There's a clearcut differentiation of love that tries to conquer and a love that's in to sacrifice. The book also throws light on how vulnerable the world has become as technology is expanding, how little an issue is capable of shaking the mankind, how little are we to trust people and how unpredictable the situations turn out to be. 

Her Voice.

It isn't so me writing something about gender equality and feminism because such concepts never existed for me in my grey matter. Having looked at the world in a very different view from how it is seen by the world, though these concepts existed since every time ago, never thought they would sometime matter to me too. 

People seem handicapped having everything perfect. Probably, having everything makes them handicapped too. One can be truly considered impaired if they have an inability to accept the fact that God created men and women to be equal. The inability to look at people without differentiating them is the greatest disability of all. She is independent? It's absolutely ok. She's making a living alone? Makes sense. She wants to stand on her feet? Do you have a problem? She'll walk alone all night? Why do you dare hurt her? Oxygen isn't reserved much for the masculine and lower for the feminine. 

From the fallen debris, raise yourself. Raise to keep your thoughts high. 

The greatest of men will come back to take his woman if she's left behind. After she's put every ounce to give a life, the greatest sin would be to leave her back. If she's weary, hold her. If she's fallen, pick her. If she's exhausted, boost her. But make sure, she's beside. That's the greatest reward you can give yourself. 

To make her win is the biggest victory you can aim to achieve. Because ultimately, we coexist. 

Review of "Into the water" by Paula Walkins

Once in a while you read a book that strikes you so deep, one like me cannot sit back and assimilate it for a while. So here's me sharing about it. Sharing is caring. Isn't it?

The back drop of the story is, there are a series of suicides or attempted murders (particularly women), the victims of which drop themselves down into a river (or are pushed down into it). The history dates back to very ancient incidents where it is believed that witches and seducers are pushed down into the river from a high cliff.

In the current scenario, a woman journalist, Nel, working on the project of covering and exploring the stories behind the deaths is herself found dead in the river. This digs the past incidences of a high school girl,  who jumped off the cliff and killed herself, reasons unknown. Also, the wife (Lauren) of a policeman, murder or suicide unknown.

The novel is written in a combination of first and third person narrative. What struck me the most is how each one of us have a different perspective of things that go on around us. How what we see and hear can strongly manipulate the truth. 

'THE TRUTH', how far does any person be sure of knowing the truth? We all assume. We are afraid of not knowing the truth. So, each one of us end up creating "our own truth", in the most convenient way that fits best for us. All we think of is how to keep ourself safe and clean. Isn't it? Is what we see with our eyes and what we hear always the "REAL" truth? Often it happens we fall into that zone that what we see gets manipulated and we only remember how we want the truth to be. The ability of humans to manipulate themselves perfectly and without flaw is the most amazing. This ability is often consequence of our inability to face the reality and inability to carry the burden of our own guilt. This perspective is not just in case of significant and big issues. But you know this thing called "TIME"? it unveils everything. It justifies every action, it makes everyone bear his own consequences. It will punish you, it will reward you, it will rise one high and it will bring one to ashes. 

The book depicted how we humans think, how we anticipate situations, how we face struggles, and how we adjust ourselves into our own truths. 

Please find space for contradictions and suggestions in the comments section below. 

Day 56: Last Day

Every journey has an end and promises of new beginning.

Today was the last day of INSc- INSA- NASI Summer Research Fellowship - 2018.

It was a wonderful journey with innumerable experiences. During this internship, I've packed for myself most of what I could grasp from National Agri-Food Biotechnology. It has given me everything one could ask for in their first research lab experience. 

My sincere heartfelt gratitude to my guide, Dr. T. R. Sharma, Executive Director, NABI, for giving me this wonderful opportunity to learn for which I cannot thank him enough. The guidance, monitoring and opportunity he's given me to learn what I was interested in, leave me indebted to him. 

He assigned me to Dr. Joy K Roy, Scientist E, whose work involved plant breeding and molecular markers. His invaluable suggestions and encouragement guided me throughout the training period. 

Thanks to my college Principal, Prof. Rishikesh Autade, who is a constant source of inspiration for me. He inculcated the habit of writing this daily blog. At the end, it feels great to get back to older posts and take a peep at how the days at the training began. Writing this blog had one big advantage. Everyday, I felt I should share something new and with this thought I would indulge in one thing or the other at the lab and tried to learn new techniques and procedures though they were not a part of my project. Also, thanks to Prof. Bhausaheb Ghorpade who is an enthusiastic learner and teacher who would always encourage me to learn new concepst and broaden my knowledge. All my college staff have in one way or the other helped me by increasing my knowledge in basic concepts.

Ms. Ankita Mishra, Senior research fellow at NABI was a true blessing to me. From the beginning, she patiently taught me experimental work at the lab. Bearing through my faults and errors, helping me learn and explaining methods and results, she provided a friendly environment for me to get adjusted to. Ms. Vinita Sharma, Junior research fellow, taught me lot of things too. Her nature of questioning every concept, searching for reasons gave me a new insight to look at science. She would often assign me little tasks from her daily work and helped me learn. Not to forget Dr. Abhishek Bhandawat, National Post Doctoral Fellow who would spark the beginning of great discussions starting from concepts of science to theories of life. Hearing these three people discuss, encouraged me to explore more and know more what remains to be unknown to me.

Other lab mates, Dr. Himanshu Sharma and Mr. Pankaj helped me acquaint myself to the lab environment and helped me clear doubts regarding various concepts.

How can I close without leaving a thank you note to my friends Silky Gandhi and Mohini Pal Choudhary who accompanied me in exploring the city and helped me adjust during the training. 

All the above acknowledgements would me meaningless if it weren't for my parents who encouraged me to take up the internship though it was really a long distance away from home. They always believed and confided in me during times even when I couldn't trust myself. I am really grateful and thankful for my family for everything they gave me and for all the things I have today. 

Not to forget my CABT friends, who rang me up often, asking my how-abouts and where-abouts. Thanks for always being there. 

There are these 4 constant viewers of my daily blog writing, who are a source of encouragement. Thank you so much. I would really like to have your comments how it felt being through in the journey with me. 

Above all, praise and glory be to the Lord, who was always there for me. 

Day 54

End of the week again. 

Continued with scoring of markers and sorted the markers based on their position on chromosomes. 

Wheat is an allohexaploid (AABBDD, 2n=6x=42) with three genomes namely A, B and D. It has one of the most complex genomes.

Evolution of wheat:

1st evolutionary event- Interspecific hybridization between diploid wild einkorn wheat, Triticum uratu (A-genome progenitor, genomic constitution AA) and diploid wild grass, Aegilops speltoides (B-genome progenitor, genomic constitution BB). The resultant hybrid was the tetraploid emmer wheat, Triticum turgidum (genomic constitution AABB)

2nd evolutionary event- tetraploid emmer wheat Triticum turgidum var. durum crossed with the diploid wild goat grass, Aegilops tauschii (D- genome progenitor, genomic constitution DD)  and led to the origin and evolution of allohexaploid bread wheat, Triticum aestivum (genomic constitution AABBDD). 

Commercially cultivated wheat are basically of two types, i.e. durum wheat (Triticum durum) and bread wheat (Triticum aestivum). 

 

Day 52:

Today,

From the results of polymorphic survey of markers and genotyping of mapping population, we did scoring of the gel images. 

Scoring is comparing each individual of the mapping population with it's two parents. The progeny resembling parent 1 will be named A and that resembling parent 2 will be named B, the heterozygotes that have inherited from both the parents are scored as H. 

This scoring is done for each individual marker that was identified to be polymorphic in the parents.

Later the data obtained, will be used for construction of linkage map.

fig. gel image of genotyping of mapping population (94 individuals) with a specific marker

In the gel image above, observe the bands of P1 and P2 in row 1 and row 3. The progeny resembling the parents or being a hybrid of both parents can be identified easily. Example, progeny 11, 13, 14, 15, 18 and 22,etc resemble parent 1, while progeny 1, 2, 3, 5, 6,etc resemble parent 2.  

Day 50:

Why do we use 70% ethanol for disinfection or cleaning microbiological work benches and not absolute alcohol? 

- Absolute alcohol or pure alcohol is a stronger solution as compared to 70% ethanol. 

Pure alcohol coagulates all the proteins in contact. So, when we use pure alcohol, on entering the cell of the microbe, it will coagulate all the proteins that are present immediately inside the cell wall and will not be able to reach the inner parts of the cell due to the formation of a protective coat of the coagulated protein. So, the inner lipids, proteins and other components remain intact inside the cell. This would mean that the cell has become inactive but it is not dead yet. Under favorable conditions, the cell once again becomes active. Consider using 70% ethanol. Since it is diluted with water, the rate at which it coagulates the proteins is comparatively slower than that of pure alcohol. So, it will penetrate deep into the cell before coagulation of protein begins. So, all the proteins inside the cell and  those close to the cell wall get coagulated eventually  and the organism dies. 

This is the reason for using 70% ethanol for disinfection and sterilization.. 

Day 49 :

A thesis or report is a crucial part of research. Every finding, research or innovation is documented as a thesis or a research paper. There is certain format for presenting this document. A clear and properly sorted revealing of the research work done, is essential so that other people in the community can understand and proceed into deeper research or extend their knowledge. 
Here's a brief format for writing a good report or thesis. 
 - A unique title: The title of the project must be precise and explanatory regarding the work done.
 -Abstract: Abstract should be short and express the core ideas behind the research. It should be a single paragraph.
 -Keywords: It includes keywords or phrases and should not contain those words that appear in the title. All the words should be separated by commas. 
 -Abbreviations: The reader should be introduced to a list of abbreviations that might be used throughout the paper. They are placed immediately after the abstract and before proceeding to the main body of the report. 
 -Main body of the report

1. Introduction
1.1 Background: In this section, the context of the research is set by giving a brief background, the research problem is discussed and how the problem will be solved is mentioned. It will include why the research is being done. This section can be sub-layered into one or more sub-sections and sub-sub sections. 
1.2 Statement of problems:  This helps the reader to understand the context of the problems, why the research is important, who will benefit, what steps the researcher will take to try and fill the lacunae or improve the situation.

1.3 Objectives of the research: This enlists the objectives of the research.

1.4 Scope

2. Literature review:
2.1 Information: This includes the works consulted by the researcher in order to understand and investigate the research problem. Precisely, this section looks at existing research that is significantly related to the current work being carried out by the researcher. here, it is necessary to evaluate the source being used and show the relationships between different works and discuss how it relates to the present work. 

2.2 Summary

3. Methodology
3.1 Concepts:  This will include how the results were obtained and from where the data or methods were obtained. The research methods must be appropriate to the objectives of the study.  Also, the problems that were anticipated and steps taken to prevent them from occurring should be described. 

3.2 Methods
This will include the details of the experiments carried out. Methods and protocols involved in the research work carried out. 

4. Results and Discussions
4.1  Purpose: The purpose of writing the results is to present the results and make them meaningful to the reader. The results should be presented in an easily accessible and understandable format. Example: in a graph, table, diagram or written text. Usually the data collected are attached in the appendix, if needed to be included.  All the diagrams and figures should be accompanied by text that guides the reader's attention to significant results. 

5. Conclusion and recommendations: 
5.1 Conclusion: it gives a summary of what was learned, what remains to be learned, the shortcomings of the work done can be exposed, benefits, advantages, disadvantages and recommendations are included.

References: This is systematically showing the sources of information or ideas that were helpful during the work. The reference section is briefly a list of all works the writer has cited or referred in the text. It should be noted that the author's name should not be CAPITALIZED or bold faces.

Acknowledgements: It will include statements of gratitude in producing the work and shall be decided by the author. 

Appendices: The appendices should be numbered appropriately 

*END* 

Universally accepted way of writing the authors: 
One author: Davis 1984
Two authors: Hamelo & Myovi 1998
More than two authors: Glory et al 2018

Source of above information: AuthorCafe. (www.authorcafe.com)

Day 47 :

Sunday again, another week passed by un-noticed.
This week was all about estimating amylose content in starch samples isolated from a F2 mapping population. It was a huge task to perform as the mapping population comprised 381 individuals. Also, at the same time, primer screening for polymorphism and genotyping in F2 progeny was done.
As , only a final week of internship remains, it's time to start preparing final report and wrapping up the things.
It seems a long way since applying for this internship, waiting for the results, accepting the fellowship offered, joining the institute, getting acquainted with the lab, getting an essence of true research, mingling with scientific community, failing and raising up, doing mistakes and learning and now with all the experiences and enthusiasm to stay in the field, work hard and work smart.

Each and every person at the lab taught me something or the other, it includes both things to do and things not to do. Every day had something new to teach and something new to absorb.
Watching everyone plan, discuss, work and learn has in it itself, great joy. In a scientific community, everyone has something to give and a lot to take from others. The most youngest in the lab might have a lot to teach to others while the seniors do not hesitate to take help. The discussions that go on at the place are worth listening to. they open the channels of thinking wider and bigger. It is really a great opportunity for having been able to get exposed to such an environment.
Having great time.
Have a happy sunday. 

Day 44:

Regarding the query: 
While plotting a standard curve, 10 mg potato starch is taken but while proceeding for samples, why do we use 20 mg of sample?

When we plot a standard, we are using potato starch which is pure amylose without amylopectin. So, when we pipette out 20,40, 60, 80 and 100 microlitre of sample, we assume it to be 20%, 40%, 60%, 80% and 100% pure amylose concentration in the series of tubes. 

In case of samples, i.e, starch, it contains both amylose and amylopectin and it may also contain other impurities. So, if we take a 10 mg starch sample, we are practically assuming only 5 mg of sample for pure amylose. It gives a much lesser amylose estimate against the standard plotted with 1 mg/ml concentration. Hence, it has been standardized to use 20 mg starch sample considering the presence of amylopectin and other possible contaminants in the sample. 

Day 43:

Working Principles of various instruments:

1. Micro-Pipette : Suction pressure

2. Centrifuge: Centrifugal acceleration, sedimentation principle

3. Autoclave: High pressure and high temperature for killing micro organisms

4. Spectrophotometer: Measuring the intensity of light absorbed by a substance.

5. pH meter: Measures the concentration of hydrogen ions present in a sample and provides a value as how acidic or basic a solution is.

6. Vortex mixer: The motor attached to the rubber surface oscillates rapidly in circular motion. The motion is transmitted to the liquid present in the tube and a vortex is created which provides proper mixing.

7. Magnetic stirrer: A rotating magnetic field is used to spin a stir bar (flea) which is immersed in the liquid thus creating a rapid spin which causes uniform mixing of the solution

8. Thermal cycler: Raises and lowers the temperature of the block as per pre-programmed steps allowing denaturing, annealing and extension.

9.UV Transilluminator: Emits high levels of UV radiation that help to visualize DNA fragments in dye stained gels.

10. Laminar Air flow: Consists of a filter pad, a fan and a HEPA ( High Efficiency Particulate Air) filter. HEPA removes all air borne contaminants to maintain sterile conditions in the LAF hood. 

Day 42: Errors and trouble shooting

In my previous blog, I have described about amylose.
Today, I'll share about steps in amylose estimation, errors that might occur at each step and how to avoid.
1. Plotting Standard curve.
Standard curve is plotted using standard potato amylose starch which is pure amylose without amylopectin. 10 mg/ 10ml working solution is prepared by:
a. Weigh 10 mg potato amylose starch. Dissolve in 1 ml DMSO (Dimethyl Sulphoxide) 
b. Vortex well
c. Incubate in waterbath at 85C for 10 mins.
d. Dilute it to 10 ml.
e. Pipette out 0, 20, 40, 60, 80, 100 microlitre of potato amylose starch into different tubes
f. Add 20 microlitre Iodine (I2:KI ) to each tubes.
g. Make final volume to 3 ml in each tube.
h. Mix well
i. Stand for 20 minutes
j. Take OD at 620 nm. 
plot the values obtained graphically. A straight line is obtained. 

Precautions:  i. potato amylose starch should be accurately weighed.
ii. vortexing properly before incubating is necessary or else, the starch will gelatinize at the bottom.
iii. Pipetting should be clean and bubbles should be cleared while pipetting.
iv. Fresh standard curve should be plotted everyday, because pipetting varies day to day and person to person.


2. Samples estimation.
a. Weigh 20 mg sample.
b. Dissolve in 1 ml DMSO
c. Vortex properly.
d. Incubate in waterbath at 85C for 15 mins
e. transfer the 1 ml sample to fresh tube
f. Add 9 ml water.
g. From the 10 ml working solution, pipette out 100 microlitre aliquot into another test tube
h. Add 20 microlitre Iodine solution
i. Make up volume to 3 ml.
j. Incubate at RT in dark for 20 mins.
k Take O.D at 620 nm

Precautions: 
i. Iodine is usually added after sample and water are added. 
ii. The reaction is light sensitive hence should be kept in dark.
iii. The incubation period should not exceed the limit because the colour complex disappears. 
iv. Iodine, sample and water should be properly mixed and should not remain on the walls of the test tube. 

Usually 2-3 technical replicates and 2 biological replicates are done.

Today, estimated amylose from 50 samples. 

Initially we used to estimate 25 samples in a single lot. The results were poor because by the time we would reach the last samples, their colour would disappear. Therefore we now carry out 12-13 samples in one lot and complete 50 samples in 4 lots. 

That's all about procedure for amylose estimation. 
Good night.   

Day 39:

Regarding yesterday's query in the comments,
As already mentioned, starch is made up of two glucose polymer fractions, amylose and amylopectin.
Among these, amylopectin is easily digestible than amylose. Digestibility is a major concern in the components of food that we eat and determines nutritional quality. At the same time, high amylose starch/ resistant starch (healthy starch) is highly preferred for preventive measures against obesity and related health conditions. So, the present study was about estimating variations in amylose content in a population that was modified for amylose content.
That was the reason for focusing more on amylose during the estimation.

More about amylose: 

• Amylose is known as a linear polymer, but is not defined as just a straight chain molecule. It frequently forms a helix and is thought to intertwine even through the several layers of amylopectin. 
• Amylose consists predominately of α-l,4-D-glucose bonds.
• It makes up approximately 20-30% of starch.
• Amylose leaches easily from swollen granules at a temperature slightly above the gelatinization temperature
• It is important in plant energy storage. It is less readily digested than amylopectin; however, because it is more linear than amylopectin, it takes up less space. As a result, it is the preferred starch for storage in plants.
• Amylose is not soluble in cold water
• It is an important thickener, water binder, emulsion stabilizer, and gelling agent in both industrial and food-based contexts

Day 38

21st June- Today CIAB and NABI jointly organized International Yoga Day at the campus. The staff and mob of the campus performed yoga asanas and started their day's work. 

   

    Back to work, today I'll be sharing about amylose estimation from starch samples

Starch is a major component of most cereals. It comprises of amylose and amylopectin. The amylose content in the grains plays a major role in digestible properties and nutritional parameters. Plant breeding is carried out often with an aim to enhance and regulate amylose content of the cereals. 

Amylose estimation is widely estimated by Iodine- starch complex colorimetry. 

1. A standard curve is plotted using potato starch which is pure amylose without amylopectin.

2. The standard curve is used to estimate the content of High Amylose Maize (HAM) whose value is already known to be 66%

3. If the HAM sample matches with the known percentage, then we proceed for amylose estimation of our samples.

4. Starch is insoluble in water. Hence it is dissolved in DMSO (Dimethyl Sulphoxide)

5. The samples are then incubated at 85 C for about 15 mins. During this process, the starch granules are burst open and the amylose solubilizes in the DMSO solution. The remaining residues gelatinize at the bottom of the tubes.

6. The amylose solubilized in the DMSO is collected into a new tube and diluted appropriately. 

7. Further, an aliquot is taken and transferred to another tube. To, this Iodine solution is added and appropriate volume is made up. 

8. The samples are kept undisturbed for 10-15 mins for color formation to occur.

9. The intensity of color developed is estimated in a spectrophotometer.

10. The absorbance values obtained are analyzed by using the standard curve plot and the % amylose content is obtained.

Day 36 : All about primers

A primer is a short strand of RNA or DNA (generally about 18-22 bases)  with a 3'-OH end that serves as a starting point for DNA synthesis. They identify sequences on the template DNA and upon providing required temperature, they anneal and start copying the sequence of the parent strand to form a new strand. Every primer has it's own annealing temperature which is usually 1-2 ⁰C lower than the melting temperature (Tm) of the primer.

• Primer Melting Temperature (Tm) by definition is the temperature at which one-half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability.

Primers that are already known to amplify sequences in wheat are used in the parents and the mapping population of the current study. 

• These primers come in vials lyophilized.
• Then they are diluted to concentration 100pMol/micro-litre. (stock) The volume of dilution is mentioned on the vials.
• Then working solution of primers is made by taking 100 microlitre of the primer stock and making up volume to 500 microlitres.

These primers are further used for screening the parents P1 and P2.

If the primers show polymorphism for the pair of parents, then they are proceeded for genotyping in the progeny. 

Iron - Cushion Man

As strong as Iron
As Supportive as Iron

Leader of the House
Builder of the Home.
Fierce from appearance
Mild on the inside.
He will never tell you
how much he cares,
how much he loves.
He will only show you.

Because, for him,
actions matter more than words.

He'll hold the umbrella
in the harshest storms of life
He'll give you the shade
under the hottest shot of the sun
Watching your step as you grow
Showing the way when you fall
All his life has passed in securing Himself
The himself in you.

Have you thanked your Dad enough this Father's Day?

He did not tell me to work hard. He showed me how it is done.