A Butterfly and Biotechnology

One year into PhD - my BTS experience!

2017-12-22T21:47:00.001-08:00

Dear readers,

I apologize that I stopped writing since last year. I don't have any great excuse other than poor planning. I was lost on my way to understand marine biology. As you might be knowing, I switched field, moved from my focus over bacteria and human disease related studies to climate change and marine biology. However, my research focus continues to be in molecular biology. Long story short, I was learning all about climate change, ocean acidification, ocean, marine animals in the past year. I even learnt how to spawn and culture oysters. How cool is it? Not cool?! Well, I think it is cool!

I can setup mini Ocean acidification systems which mimics the future acidified ocean conditions; I even gained some plumbing related skills. This was totally a new experience and I thoroughly enjoyed it. However, I had a tough summer in a rural fishing village in China, learning how to grow oysters from the Chinese oyster farmers (for those who do not know what I am doing: I am doing PhD at the School of Biological Sciences, The University of Hong Kong and I went to mainland China from Hong Kong Special Administrative Region *SAR* for performing experiments). It was such an awesome and scary experience at the same time. Travelling to a remote village in China which is not possible for any common traveler or tourist to go to, without knowing Mandarin, was a mind blowing experience. To give a brief view of how this village looked - imagine a street, one street near sea side, and there were only two restaurants, and the food they served was very different from my taste (Wait, don't imagine fancy stuffs, this was NOT FANCY at all). Okay, fine, enough background, here we go, I ramble about my one year PhD experience...

Importance of language and communication for mental health

From my China experience, I realized how important communication is for the mental well being of humans. Imagine, you are in a place where nobody speaks your language, you can not communicate to anyone, neither talk nor understand their language, also added (dis)advantage, you can not read any signs as everything (buses, shops, most importantly the menu cards in restaurants! :( ) is written in Chinese. Literally, you need to rely on your "own version" of sign language which the locals might not understand as the signs that you use might totally mean something else in their culture!

A picture of the room in which I grew oysters.
Location: A village ( I really do not know the Chinese name)
near Zhanjiang, China.

I was thinking it would be easy to survive in China as I already managed to live happily in Hong Kong for months. I did not think that not knowing the language would affect me mentally, but it did! Thanks to Michele, my friend and lab-mate, who came with me to China to perform field experiment. She can speak, a little Mandarin and Cantonese which the locals understood. She helped me in communicating (translating) and she was the only one in that village, that I can talk to! But, imagine I can not always talk only to her, I wanted to talk to the locals as well, and it was never possible.

In the first few weeks, my brain got so confused as it could not make sense of what people are speaking around me. I started feeling like "I am so dumb" that I can not understand the language. In the first few days it was so stressful as everyone around me was planning to setup the experiment, while I just stood there not understanding a word of what they are planning. I even made some mistakes due to communication gaps and misunderstandings. All these mistakes, made me feel like I am worthless of doing anything, and depressed.

Tip 1: Communication with your PhD supervisor should also be top priority.

I slowly started adjusting to the place, Mandarin did not sound like an alien language anymore. Surprisingly, sometimes I was able to partially understand the context of the conversations happening around me. Now, I know few words in Mandarin like "Chifan - Eat (very important :P), siasia - thanks, dhuve - yes or okay, shuve - water, hai shuve - sea water, moolie - oyster, ma - at the end of a sentence is a questioning word". I know few more words, but "hello, this is not a how to speak Chinese blog post".

Blood, Sweat and Tears! BTS!

(No, not the Kpop band or any other music band)

Being from India, I can not eat "just boiled" vegetables or meat without any added spice! I mean, there was some sauce and chili added, but that was not enough flavor for my Indian tongue! In the first few weeks, the lady in the oyster farm (where I lived and conducted experiments) cooked really good food, as she was cooking for "foreigners" for the first time, she put so much effort. I enjoyed the food for the first few weeks, however, adding oil to my language problem, the lady started to "just boil" the veggies and meat after two weeks (or at least it tasted so to me).

It was so depressing that I can not speak, can not eat good food, but need to do so much physical work to feed my oysters and grow them. I literally needed to feed my oysters several buckets (approx 20 liters each) of algae twice a day. Until this point in my research life, I was this girl, who used to work wearing a fancy clean "white" coat in molecular biology lab, never used to doing physical work. Time changed, and in China, I was, far away from home, doing physical labour work, everyday, changing water and feeding my animals when I do not feed myself proper food. This work was so depressing for me, it was such a good experience, that I can closely relate to people who do tough physical labor on a day-to-day basis and empathize (I know, I talk about being depressed, and contrast, talk about good experience in the same sentence). How difficult it must be for them! After all, growing oyster was an important skill that I needed to learn for continuing my PhD research.

Everything in life happens for a good reason after all, the trouble we go through today will definitely aid in molding our better versions in future.

I realize, months after returning back to Hong Kong (HK), "I am really capable of adapting to any extreme living conditions". Oh wait, did I tell you, the living condition and infrastructure at the village was also not great! There was sudden typhoon, heavy rain and adding to the sad story, we (Michele and me) both got Athlete's foot infection (I never knew about this infection before in my life) as we were always working in sea water. If you thought the athlete's foot infection occurs when you are literally so dirty, it is not the case! One can get athlete's foot infection if one is continuously working in infected water areas. The workers in the farm had the infection, and somehow we managed to get infected from them, thanks to all the heavy rains and sea water! Our infection was not advanced stage like the ones you find in "google search results" (I know you googled it, no? okay, go now, google and come back here), ours was starting stage. Luckily, we took care of our infection in the early stage by washing our legs with Vinegar, keeping our foot dry and by wearing water boots whenever we stepped out. I spent some nights, late nights (say 2 AM), changing sea water for the tanks in which I grew my animals (I do not understand why there was sea water available only during odd hours in that farm, something to do with the tide levels, but I do not understand why they couldn't store the water!), all dirty soaked in sweat and sea water, really depressed, crying to sleep. It was truly a blood (the infection part :P), sweat and tears experience!

I survived my first year!

After all the hard work, I collected the samples required successfully and left happily back to HK. Also, took a nice one month break after summer and went back to India. I processed some of my samples, and successfully passed my probation seminar as well. Now, I totally understand why everyone was saying "doing a PhD is not easy". Yes, it is not easy, sometimes you might doubt if research is really your passion when the time gets tough. Yes, it can be really depressing even when you are working on your dream job, but,

"hey, success does not knock our doors on a Sunday morning, it comes to people who are ready to experience - blood sweat and tears!" - Kanmani (haha, I always wanted to do this)

It is okay to cry (which I do all the time, does not mean I am weak!), it is okay to take a little step back, relax and rest, but, it is never okay to give up! One lesson I learnt in my first year PhD is, work is important, being passionate is more important, but never at the cost of my happiness, family and personal life. I learnt to work, not to impress anyone, but just to satisfy myself and my curiosity. Striking a balance between research life and personal life is what I learnt during this one year journey. After being away from home (India) for a year, I realize, I value 'people in my life' more than anything. However, there is no compromising on the career path, I am so greedy that I want to be happy and successful both personally and professionally.

Tip 2: Avoid things that make you sad. Paradox: sometimes working towards your passionate goals can also be tough and make you sad, don't give up during those tough times!

I hope next year, 2018, is a highly productive and successful year for me; for you too. Let us all work towards our happy lives! Happy new year 2018!

*For fun*

*New year resolution: #1 Write here in my blog more often*

*You know what was my last year new year resolution? To brush twice a day :P Serious, it is so difficult to brush when my bed calls me to cuddle, but I did well this year, I brushed almost everyday twice, except two or three days :P *

Sea Butterfly!

2016-10-17T08:41:00.000-07:00

Neih hou!

Don't roll you eyes wondering what it is! "Neih hou" is how you say "hello" in Cantonese. Guess what, I am in Hong Kong and therefore the language Cantonese. I came to Hong Kong this summer as an intern before officially joining as a PhD Student. This is my second experience abroad, far away from home, new language, new culture, I expected me to have a "really" bad cultural shock, but, actually I experienced only 50% of what I expected. For a girl who have used the marina beach only for eating "sundal", bikini in beach was a shock (FEEL FREE TO JUDGE ME!). The first time, I went to the big wave beach here, I was the only one who was totally covered, when everyone was enjoying their Beer, I was slowly sipping my lemon juice (THE ODD ONE OUT!). I realized how beautifully different the world outside is! There is no single standard for right or wrong, it varies in different countries, in different regions around the world. And it is all right as long as you don't hurt anyone and don't force anyone to do something. I am learning to appreciate different culture and perspectives.

Life is beautiful! But, also there are a few "mean" people everywhere! - The UNIVERSAL TRUTH

I always wanted to travel abroad and study, see how things work in other countries, understand how people do research in places other than India. I am a person who loves home food and wants to stay with family, at the same time. And when I weighed the comfort of staying at home over doing research abroad, the later weighed quite heavy, so, here I am in The University of Hong Kong (HKU)!

Did you miss my first flight? Don't worry, go "here", read and come back here :)

I always wanted to learn new things and was curious. I have worked so far with microbes, especially bacteria. I know basic molecular biology, genetic engineering techniques and always wanted to work at molecular level. When it came to PhD, I looked for a challenging, new, interdisciplinary area where I can learn exciting things and I found marine science perfectly fitting all my specifications when I was browsing through various research topics. Especially, I feel the "excitement" part is very high in this particular field because of the field trips into the sea for getting samples and also Hong Kong consists of 263 islands, making it a perfect place for doing marine research. HK is also a global financial centre after New York and London.

Let's have a quick glance of the interesting tit bits that I learnt in HKU!

1) First encounter with oysters!

I am this person who have never seen oysters before in my lifetime, but, in pictures. First day, I go to lab, say "jousan" (good morning) to everyone and also to oysters. The very first thing I learnt once reaching the laboratory was how to take care of oysters, growing them, feeding them, cleaning them :) Wah, it felt great, because, it was so lively, though the oysters don't swim around like fishes. Obviously, you need to sacrifice them later for performing experiments, but still, I found it new and exciting!

Oysters are filter feeders, oh yeah, they filter the water! You are right! Go, remove the filter from your fish tank and throw some oysters inside it! Generally, they feed algae to oysters, depending on the stage of life cycle in which the oyster is in, the type of algal feed varies. Example, small sized algae for the baby oysters. (Algae used in my lab: Isochrysis galbana)

2) Strip Spawning!

Spawning - are you wondering what does it mean? Even I was wondering the first time I heard the word. It is a process by which eggs and sperms of the oysters (or in general, any marine organism) are released into the water and then they fertilize, and you know what happens after that!

Strip spawning is the method in which you remove the eggs or sperms from the animal carefully, oysters are hermaphrodites, it means, they have the ability to switch their sex, they can be male or female. They change their sex depending on their life cycle. During strip spawning, we usually, puncture the gonads, take a tiny bit of gamete, place it on a microscope to check whether the gamete is male (Sperms) or female (eggs). Based on what gamete it is, we separate and count the animals as males or female, and, collect their gametes separately for fertilizing them later.

While doing strip spawning!

3) 2D Gels!

I have learnt the basics about two dimensional SDS PAGE before, but, never had hands on experience. I learnt doing it, was fun! I find myself better than my undergrad version who was all nervous and shaky when entered a new lab! I learn new things with confidence and curiosity.

4) SWIMS!

I spent my summer in the Swire Institute of Marine Science (SWIMS), which is located far away from the bustling Hong Kong city. The cool thing about this institute is - it is in the shores of the only marine reserve in Hong Kong and it is really a great pleasure to live, read and research in such a beautiful place, far away from the city means far away from all kinds of pollution. But, obviously, you will starve unless you have some culinary skills, other option, you can buy and stuff your fridge or survive with instant noodles. But, it is really a good place for a marine scientist to do summer research in HK.

5) HKU!

For a student who did her undergrad from a small college in Sivakasi, where there were "millions" of rules (yes, literally millions of rules) to say what not to do, HKU is like heaven (But still, I also like my teachers from the previous colleges, they were the ones who taught me the basics and kindled the passion for science in me, they are my first source of inspiration). Free learning - there is nobody to force you to do anything, you are free to choose the way you want to learn, of course, there are some rules! The 'on campus' restaurants and canteens in HKU appeared like "5 star hotels" to me comparing the university canteens back in my college. Every time I eat my meal here, I feel so blessed, it urges me not to waste food (Doesn't mean you can waste food if you eat in a normal place! Don't get this wrong!) which several others don't get in this world. This is more than what I expected.

The laboratories in HKU are well equipped, the quality of research is great. This is why, I exactly wanted to go abroad, to see, how the world out here is so different from my country. There is some workshop or seminar or symposium going on everyday, and, if you want to learn, you are free to join in any of these.

I am learning so many other things than just research and science. Things for life, reasons for being responsible and above all I realized, doing PhD is not that easy, but still, I am happy I chose this tough path, there would be no fun in doing something very simple, right?

6) Writing a paper!

I have never written an article for a scientific journal before, though I have experience of scribbling here in this blog :D Now, I am learning to write a research article for journals, it's so exciting and fun to learn new things. I hope I will soon write an article about "my first experience in writing an article for scientific journals" once I finish writing my first paper.

Reading papers, reading more and more papers, learning new things is the main part of my PhD life. I hope, at the end of PhD I will just not get a degree but evolve as a better person. Share what new thing you learnt recently or your experience in the comments :)

And yeah, I named myself as SEA BUTTERFLY, now :P

Care when you CLONE!

2015-10-17T03:47:00.000-07:00

Hi there! (am using Whatsapp) :D

When I started writing this, I wanted to start with a "Hi" and suddenly, the famous Whatsapp status came into my mind! :P See, am really addicted to Whatsapp nowadays, changing DP and Status often. Oof, I should stop writing whatsapp status and start writing here. (This is NO ADvertisement for Whatsapp :P)

In the last post, I said that I'm in the USA doing research and now, am back in India after successfully completing the internship! Yaaai, back home, back home! It feels good when you are in home, yes, am happy to be home. But, sometimes, its good to visit places and learn new things, leaving home. Let's come to the purpose of this post now! In this post, am gonna share few things I learnt from my internship.

I was basically cloning different fusion proteins and was doing invitro degradation studies of those cloned proteins using western blots. I've done cloning before, when I was doing my under graduation (UG), but, I think I've grown up now to understand things better! Things once I found very difficult have become easy now! I'll give you an example: During my UG, I found it very difficult to cut out the required DNA band from agarose gel for gel extraction. My hands use to do "Disco dance" when I use to try cutting the gel. (I think, many students suffer from this "shaky hand" problem! Don't worry, you'll be alright soon) But, now, guess what? Am doing it like a BOSS! :D I think, it comes with practice or should I say, "I've grown up!"

Back to the point, when I was trying to clone an insert into a plasmid vector, the first time, it was a failure! I got only the vector without any insert. And, then I did trouble shoot the strategy and found out a correct way of doing it, and, in the next attempt, EUREKA, success! :D

Here are few things you should care while cloning (plasmid vector, E.coli):

1) Always try to use two different restriction enzymes

Using two different restriction enzymes will help in avoiding the self ligation of the vector i.e., when you are restricting the vector with only one restriction enzyme and then purifying it, using it for cloning, there is a possibility for your insert to get in the vector but also there is every possibility for your vector to self ligate!

Self ligation of vector

2) Do Gel extraction purification whenever possible after restriction

This is where I did mistake when I was cloning. I restricted my vector with two different enzymes, but, I didn't do gel extraction, instead, I did PCR cleanup! PCR cleanup can generally remove upto fragments of 100bp but not the fragments more than 100bp. My vector after restricting with two enzymes, gave out a fragment which was greater than 100bp. As the PCR cleanup was not efficient to remove this fragment, it got re-ligated when I did ligation with my insert! (See picture) The possibility of the actual vector fragment to self ligate is more than that of the insert.

Obviously, doing, gel extraction and purification after each restriction will give you less amount of DNA, but, trust me, even if you have 1 or 2ng/micro litre concentration of DNA, that would be pure and you'll get a good number of positive colonies at the end.

3) Restrict overnight!

Normally, this won't be necessary if your enzyme quality is good. But, I had problem with one of my restriction enzymes when I did 1 hour incubation and hence I decided to do overnight restriction. The overnight restriction at 37 degree Celsius worked fine and gave good number of positive clones at the end.

If you are doing overnight restriction then, it is better to seal your tubes with parafilm to avoid evaporation of the buffer (There are chances for the buffer to evaporate slowly and thus increasing the buffer concentration). There is also possibility for star activity (non specific restriction due to varying reaction conditions) but, even if there is star activity, you will ultimately have properly restricted product of interest also in the mixture and this product can ligate properly with your vector.

4) For mutations!

I also did point mutations in the insert, and, I hope it is better to do the mutation after getting the clone, i.e., put your insert in the vector, confirm the sequence and then do the point mutation. By this way, if you need to compare both the native and the mutated protein, you need not do two different cloning, instead, you can have a clone of native and from that you can take some amount of plasmid and do point mutation in it using mutation PCR.

I initially had a doubt, "whether it is possible to do PCR of whole plasmid with the insert, around 6Kb, using PCR?" Later, I found the answer as, "Yes, it is possible to do!". I used Pfu DNA polymerase for the PCR. Pfu is superior than normal Taq DNA polymerase for it has proof reading activity also, hence, less error! The primers which I used for this mutation PCR of plasmid was about 40bp in length.

And, it is necessary to do digestion with DpnI after the mutation PCR. The template plasmid what we are using for the PCR is generally the plasmid obtained from dam+ bacteria, .ie., this plasmid is methylated. We should restrict the PCR product with DpnI enzyme which selectively restricts the methylated DNA, providing us only with the mutated product.

Hope, this piece of information helps someone in cloning. Happy cloning :D All the best!

The Butterfly flew for the first time!

2015-06-26T11:44:00.000-07:00

Hi dear sweet lovely reader,

Didn't meet you for a long time... Just after complaining about the stipend problem in the last post, I actually wanted to write a lot here, but, couldn't find time (Such a lazy girl, I am!). As you might be knowing, the author (me) is the butterfly here :P And, I flew for the first time!! YEAH! I FLEW!

I flew like a butterfly!

It was an awesome experience flying for the very first time! (ow, no! I dint put a trans-gene in my body and fly after getting a pair of wings, but with a passport and VISA). I'm this girl, full of dreams wishing to do "this", "that" and "all kind of stuffs that one can do". And, one among those dreams was to fly one day! TO FLY FREE!(I mean, not free of cost :P, but liberation) I always wanted to fly in those big, big airplanes, but, I always have avoided that mode of transport. Reason? Obviously, it would burn a big hole in my dad's pocket (actually he can afford if I wanted him to) and I dint want that! I wanted to travel by plane with the money I earned! So, I never have traveled by plane in my past life time of about 21 years (before June 16, 2015).

And with the dream of flying and learning things abroad, I started applying for International Fellowships, the fellowships which allow students to intern abroad for two months (or 8 to 10 weeks) by paying them attractive stipends and air tickets! Once such awesome fellowship offered by the joint venture of IUSSTF (Indo US Science and Technology Forum), Department of Biotechnology - India and Winstep Forward is the prestigious KHORANA FELLOWSHIP!

This year, 2015, about 50 Indian students from life science background were offered with the fellowship and the butterfly (yeah, it's me :P) was one among the 50! When I sent my completed application just two or three days before the last date, I had hope that I would get the fellowship, I felt very satisfied on seeing my completed application (Felt like, "This is the PERFECT APPLICATION ever :P"). But, my parents were not happy with the idea of flying all alone to an alien land (That's what they think). For them, I'm still a little KG going kid, who doesn't know to wipe the snot coming out of her nose :P. I told them initially that I would never get such a prestigious, super duper fellowship. "But still it is worth trying", I said and sent the application.

The application procedure was very, very simple. Just email all the stuffs they have asked for in a single pdf to the mentioned e-mail ID. That's it! Done! For all the application and program details refer "here" or the IUSSTF website (Ask the Google God, he'll light up your path). The applications are generally open during the month of November.

The application contains portions like Statement of Purpose (SOP) where you actually should write, "what you would like to achieve through this fellowship and why you would like to go abroad for internship", kind of stuffs. In my point of view the SOP should not be very scientific, but, still there are many people who write it in a damn scientific way like a project proposal! I never did that way or will do! You know, I even quoted this blog in my SOP to showcase my interest :P :D

So, the next thing in the application is your "academic record". There are people who say that your class 10 and class 12 marks are of no use. But, I've realized that in every job application or internship application or any application, the important thing asked for is your MARKS starting from class 10! So, its good if you have at-least 85%, I think the eligibility to apply for this fellowship is 85%. (I'm not sure, kindly refer the details in the application instructions or eligibility). I had around or above 90% right from my Class 10 and I think that kind of helped me in getting fellowships as it shows that am a constant learner?!

And, the other thing is your previous research experience and techniques you are exposed to. You should write about your previous research projects. I've already worked as Summer Research Fellow of Indian Academy of Sciences and I wrote about the project I did in that fellowship as well as my final semester UG project (I was a first year Post Grad student while applying for the Khorana Fellowship). So, even if you have did a week industrial training, mention the techniques you learnt during the training. (These things will help them understand how much you know).

Then, you need two recommendations from your professors or mentors who have guided you or taught you in college or during your internships. This is for understanding what kind of student you are, your plus and minus. If you really are an enthusiastic student, obviously, you would get a good recommendation from your professors. This will also help you a lot. (Make sure you get it from the professor who knows you well, don't get from some random professor who is your distant relative for he would give a good recommendation.) I actually got one from my Faculty adviser for the first year PG program and one from my UG Professor and project mentor.

Ah.., I think that's all, nothing more important about the application than the above explained stuffs.

So, I sent the application and was waiting for the result. One fine day, an email arrived in my inbox :)

It said "you are provisionally selected (so, it's not yet confirmed) for the prestigious Khorana Fellowship". I couldn't believe, I called my mom and told her and guess what was her reaction? Happy!?? No, SHOCKED! She was like, "OMG, you got selected and you have to fly alone to that alien land??!!". Yeah, she was happy also, but, very much worried also about how I would manage! That too, I've never traveled by plane before!

Then, I told my two or three best friends, not everyone, not even my classmates. The email I had received also said that as the next step I should choose three mentors from ANY lab in USA (and that was a wide choice to select) and send them (the fellowship in-charge people in India) the list of three professors whom I want to work with. And it is not that easy to select a mentor, first of all, you should find a mentor working in your area of interest and just you selecting the mentor is not enough, actually the mentor should select you :P They had a list of mentors, who hosted Khorana Scholars previously, in the application website. I tried sorting down from that list and it was really difficult, but I still managed and sent email to three professors first. Reply email arrived immediately from one of the three professors saying, "I'm on summer vacation and will be back after a month..." (yeah, it was an automated reply) and the other two dint reply even after a week, may be my mail went to their spam box? or they dint check email at all? I don't know!

I actually the sent the name of those three professors to the fellowship in-charge people in India. Again I emailed them (fellowship in charge) asking whether I can try emailing other professors as the three professors I initially preferred dint reply back and they said, "yes".

So, from the next day I started sending emails, like, one professor - one email in a day! I sent emails requesting to work in their lab during summer as a Khorana fellow; many dint reply at all, few replied that there was no vacancy in their lab and finally one professor replied, "YES". And that was one of the happiest days of my life, really, after losing all hope that I would find a mentor, his reply was like a sweet rain in a hot summer! I started jumping and dancing! Forwarded the acceptance mail immediately to the Khorana in-charge in India. And after that, everything went fine and fast. The IUSSTF is just awesome that they booked my air tickets and helped in every way possible to plan my travel. I did the DS2019 form (required from the US institution to apply for a J1 VISA) work via email (I used the email service to the fullest during these days, sending emails almost everyday). It took around a month to get the DS2019. Then the VISA procedure. J1 VISA normally takes 20 days maximum but in certain cases can take up to a month. I got in 15 days approx.

The sad part is, I couldn't go for the orientation program for Khorana and SN BOSE fellows in Chicago as I had my semester exams during that time. My ticket was booked from Chennai International Airport in Lufthansa around 2 am, June 16, 2015. I've never seen an air plane closer in my life, never seen airport also! But, that day, my parents waved me "TATA(Bye)!" in airport, I couldn't even say "bye" properly, as I was very nervous! But, I managed somehow in my approximately 35 hours journey. Everybody on my way, helped! The itinerary was like from Chennai to Frankfurt (Germany, I never knew before that Frankfurt is in Germany :P) then from Frankfurt to Chicago. Then Chicago to Lansing.

Oh, forgot to tell you, I'm right now in Dr. Heedeok Hong's lab, Department of Chemistry, Michigan State University, East Lansing, Michigan State, USA :) :) doing my internship. I'm doing protein work (chemical biology) here. It's awesome; this is the best way to spend your summer. So, if you are a UG or PG student in Life Science from India, don't forget to apply for this fellowship, this November. :)

Ahn, one more thing, if you don't have a passport, apply for the passport also, while applying for the fellowship ;) (I did that way as I dint have a passport, yeah, I was really confident to get the fellowship).

Need any help regarding the application or doubts? Mail me or drop a comment.

I've written this hoping that it would help someone who is applying for the fellowship next time. Thank you so much for patiently reading till the end of the story. All the best :)

See you soon, dear reader.

Avidadham'15

2015-01-18T02:02:00.000-08:00

Hi readers,

I got an idea! (Bulb glows bright!) Yes, an idea to meet all my blog readers if you want to! How cool! What? Are you asking me to stop!? Oh? yes? Are you asking me, "What is the relation between this IDEA and the title of the post AVIDADHAM'15?". Wait, dears. There is a correlation. This idea to meet you all is a function of Avidadham'15! To put it in a mathematical expression :

Idea = f(Avidadham'15)

:P :D This is the result of doing so much Math for semester exams. Okay, coming to the point, What is avidadham'15? Avidadham'15 is an International Conference organised by the Department of Biotechnology, Anna University, Chennai. I think, now you might have got the point. Yes, I'm a post graduate student in the department and I welcome you all to Avidadham'15. It is for 3 days! It will be fun for all research and science lovers, so, all my bacteria lovers, come and join us!

You can meet international and national scientists, present your research work in poster/oral presentation events, attend the workshops and last but not the least, you can also meet me! (I got your mind voice saying, "Come on, Kanmani is not a celebrity to meet, just a blogger!". Oh yeah, but, if you wanna meet me,just, in case :P) (One more point: The food provided in Avidadham will also be delicious :P)

I can assure you one thing about Avidadham as I have also attended it as an undergraduate student, as a participant. I learnt so many things, I got all excited and the speakers were brilliant! So, I can assure you that you would learn so many things and get a good exposure to recent research trend. Don't worry about accommodation and other stuffs, we will take care of all those. Hurry and register!

Now, as a part of Avidadham'15 team, I welcome all my blog readers and friends to the conference. Let's listen, share and enjoy the fun of Biotechnology.

Tired of the routine schedule? Searching for a platform to showcase your research work? Looking for an International conference related to biotech in Chennai? Undergraduate? Post Graduate? JRF? SRF? Professor? Scientist? This is a perfect place for you to land! All are cordially invited!

So, Coming? To meet me? Leave a comment or an e-mail! Cheers, Let's meet! :D

For more details about the conference, visit : Avidadham.org

Stipend story of a scientist!

2014-11-21T08:32:00.000-08:00

Hi readers,

It feels so good to write here, after a long (so long, in fact) gap. How are you all doing? I’m doing great, did you know, I have now joined M.Tech (Biotechnology) in Anna University, Chennai. And, after 20 years of life under the warmth of my parent’s wings, I have finally managed to spread my own wings, out of home. Yes, I have come out of my kutty (small) town, to a big city – Chennai. It feels good in one way but the other? No! It’s not feeling so good the other way. After coming here, I have realized that I was away from the so much polluted world for 20 years. This place, because of tremendous industrial development and stuffs, is polluted extremely, that you should cover your head, face, hands, foot, literally, all your body parts except eyes to keep you clean and safe from the poisonous, dusty, dirty air! Ah, forgot, you should wear big coolers to protect your eyes too. Sometimes, it feels like there is not more than 10% of O₂in the air! Villages, towns – Rural areas are heavens to live in, but, I came running to this city to achieve something; to breathe some dirt; to experience how it feels really to be independent and motivated towards a goal; and, to go crazy; to study like I have never did; to feel, try and learn everything under the sky; to make a new episode of my life!

It’s very challenging in India, especially for a girl from a small town, for a girl from an orthodox background to get out of home and go behind her dreams. After facing, so many distractions, “get married” advice, I finally convinced my parents that I would like to get out of my town, away from their warmth, avoiding their spoon feeds. I proudly was explaining them that how I cleared the entrance and how much stipend amount I’ll be getting every month, so that, they need not take care of my expenditure anymore! I was building so many beautiful castles in air with the thought of getting stipend and spending it for my food, shelter, clothing, books and if possible a small tour. Wondering, how much is the stipend money? It is just 8000 INR :P and it WILL NOT be sufficient even for basic needs if I’m spending it, without being a little stingy! And, yes, I was planning so much with that 8000 INR.

Do science!

At last, convinced my parents, came here, joined. And, the best part of the story starts here. It’s been 3months since I joined M.Tech, and, I had not received stipend, even for a single month! So sad, right? First month end, I asked my dad, “money”. He dint ask about the stipend, but, I felt so much embarrassed and started explaining him that I would get 16k altogether, at the end of next month. He said, “It’s fine, even if you don’t get it!” But, it dint feel good for me!

When we (all poor students of my class) asked about the stipend in the university department, they said that they dint receive the funds yet. I don’t know, what the problem IS, but, it is JUST not right! Every other student in my class, having left their home town, having travelled so long, from all the corners of the country, are just SO MUCH worried because of this. We know that we would receive the stipend money for all the months, for the whole years, totally around 2lacs, for sure, without fail, but the question is, “WHEN?” The stipend is meant for supporting the student’s expenditure, but, what is the idea of giving it after a long… long… gap? It won’t be of any use if we receive it, after completing M.Tech, after finding a job! We need it now; we need it to take care of ourselves and to proceed with our research. And, I seriously understood the reality why every Indian student wants to fly abroad for doing MS. At least, they would get the stipend on time, there!

Money!!! Stipend! :(

If this is the case, YOU people, who are in charge of this stipend amount, don’t announce or promise that we would receive a monthly stipend! I know, writing here, will not move a brick, but, still, just to vent my frustration out!

Sorry, for venting out all the dirt over you. You researcher, just GO, run your gels, heat up your PCR (I don't know why, but I really, love this guy) machine, work, research, toil, not to receive anything out of it, but satisfaction of going behind your dream. After all, I’m happy that I’m doing what I love! You too? Yaai, Cheers to us! Got a doubt? About the "scientist" mentioned in the title," It's me :P"

Yes, lets do it!

But, look around, life is so much beautiful with big red roses all around and very little, less thorns! What do you say?

Real Time PCR (qPCR)

2014-01-16T05:52:00.001-08:00

I don't know why, but, I really love this guy! :P Yes, he is PCR! You ask me anything, like, what technique we can use for introducing mutation? what for diagnosis? I'll answer PCR for all! :P Let me talk about him for sometime here.

PCR (Polymerase Chain Reaction) is used for amplifying any gene from a given sample using Taq polymerase, dNTPs, Primers and buffers. After PCR the amplified product or amplification is generally checked by running an Agarose gel electrophoresis. But, in case of Real Time PCR there is no need for running gels as the progress of the PCR is monitored online with the help of fluorophores. Generally SYBR Green dye, Taqman or molecular beacon probes are used for Real Time PCR. In SYBR Green method, the fluorophore binds with the double stranded DNA and produces fluorescence, and hence as the amplification increases, fluorescence increases, but the fluorophore has no specificity and hence, even if the amplified product is not the product of your interest you would find increase in fluorescence. The TaqMan and molecular beacons are probes which bind which are labeled with fluorescent dyes at one end and quencher at other end. When the fluorescent dye and the quencher are at close proximity there won’t be any fluorescence but on amplification the fluorescent dye and the quencher gets apart thus causing increase in fluorescence. These are specific to the gene amplified and hence we could monitor the amplification specifically online.

Another method is LUX (Light upon eXtension) in which the primer is labeled with fluorophore at the 3’end. The primer remains in a hair pin like structure initially, and, at that configuration it emits less fluorescence. When the primer becomes linear single strand, the fluorescence increases and when it gets extended and becomes double strand, the fluorescence increases further. As the primers are specific, the fluorescence increases only when the amplification is specific.

Melting curve analysis is generally done for the PCR products in case of LUX and SYBR Green as the fluorescence could also be due to the primer dimer. In the melting curve analysis, the product is heated thus allowing it to unwind, the melting point temperature (T_m time taken for half of the sample to unwind as single strand) is noted. The melting temperature will be different for primer dimers and the product as they differ in length. And hence, fluorescence formed could be exactly correlated only with the product after this analysis. Many of the light cyclers have inbuilt option for performing this melting curve analysis.

It has various applications; few of them are listed below:

Diagnosis where genes which are specific for a particular species can be targeted and amplified, if the amplification is positive then the sample could be diagnosed as infected. The specialty of qPCR is that it also gives exact severity of infection.
Microbiology – for comparing the differences between different species, sub species and serovars of microbes
Research - for works like cloning amplification of a particular gene is essential and also for measuring expression of a particular gene qPCR can be used.

Hope, you also loved him! ;)

Knock Out Mouse (KO Mouse)

2014-01-14T08:04:00.002-08:00

Knock Out mouse is a genetically modified mouse in which a particular gene is removed or inactivated. As a particular gene (which encodes for a particular protein) is inactivated, the mouse shows changes in external features or physical and biochemical characteristics. The first recorded knock out mice was produced in 1989 for which Martin Evans and Oliver Smithies were awarded Nobel Prize.

The various reasons for producing knock out mice are

· To understand the function of a gene

· To understand various diseases which are due to alterations or mutations in genes

· It gives an idea for understanding and treating various diseases

· To develop and test various drugs produced

Some of the diseases which are widely studied using mouse models are neuronal diseases like Parkinson’s disease, Alzheimer’s disease and various types of cancer are also understood with the help of KO mouse models. Besides these, KO mice are also used for studying heart diseases, diabetes, obesity etc.

For producing knock out mouse, a DNA fragment is designed such that it contains some random sequence in place of the gene to be knocked out with flanking sequences similar to that of the sequences which are flanking the particular gene (to be knocked out) in the original genome. Moreover, the DNA fragment also contains markers to detect the effect of knock out or Embryonic stem (ES) cells are generally used for knock out, the ES cells are collected and transfected with the DNA fragment designed. Homologous recombination occurs resulting in knock out and the cells which are knocked out are transferred back into the blastocyst, then the blastocyst is placed in the uterus of a surrogate mother (mouse) which gives birth to knock out mice.

Why knock out “mouse”?

The next question that comes into our mind is “why mouse? Why not other species?”

The reasons for using mouse as a model for understanding human diseases and genes are the similarity between the genome of mouse and humans to a greater extent. And also mouse is easy to handle.

Limitations:

There are also limitations in producing knock out mouse. Most of the knock outs are lethal so the knocked out mouse cannot grow up and survive for a longer period. It is difficult to produce a knock out mouse as there are always possibilities for random or non homologous recombination. Producing knock out rat is difficult, and the first knock out rat was produced in 2003, 14 years after the production of KO mouse.

Isolation of monocytes from PBMC (Peripheral Blood Mononuclear Cells) - Principle and protocol

2013-10-28T10:34:00.001-07:00

Whenever I'm made to realise that I'm not clear enough or good at something, I try to make myself clear with it. It happened today, during my laboratory examination, I was asked to perform monocyte isolation from a given blood sample, but, unfortunately, I was not very clear with the principle behind it.(but, still I managed to complete the experiment as I know the protocol, but, knowing the principle behind each step of the protocol clearly is very important, isn't it?). But, nothing is wrong in it, I made myself clear with it now. That's good, right?

So, let me share with you some basic principle and protocol for isolating monocyte from blood sample.

For isolating monocytes, initially we must isolate PBMC (Peripheral bood mono nuclear cells) from the blood sample. Here, let us make few terminologies clear before starting with the principle.

Peripheral blood sample - It is the blood sample obtained from acral areas of body (in general, it is the blood collected from hands by your doctor for doing blood test).

Peripheral blood mononuclear cells (PBMCs) - It is the cells which have a prominent round nucleus which is present in the peripheral blood sample. Note, all the blood cells have a single nucleus, i.e, mono nucleus, but, neutrophil, basophil and eosinophils have lobed nucleus, i.e. they are polymorphic. Lymphocytes and monocytes have round nucleus and they are referred as PBMCs.

Ficoll hypaque - It is a polysaccharide which is soluble in aqueous solutions and widely used for performing separations based on density. Here, we can isolate PBMC's from RBC's and other blood cells based on the difference in their density.

Percoll Gradient - Percoll is silica particles coated with PVP (polyvinyl pyrollidine) and preferred for biological isolations because of its characteristics like non toxicity and low osmolarity. Besides it's use in monocyte isolation it is also used in sperm selection in assisted reproduction where healthy and active sperm cells can be selected using Percoll gradient.

Isolation of PBMCs

Protocol in short - (if 1ml of blood sample is taken, dilute with 1ml of 1X PBS before starting, when you use 10XPBS it is reported that the separation is not efficient, problem occurs while overlaying ficoll hypaque)

Take 2ml of ficoll hypaque for isolating PBMCs from 4ml of diluted blood (2ml blood diluted with 2ml of 1X PBS) in a falcon (always maintain 1:2 ratio of Ficoll and blood sample). Overlay the Ficoll hypaque with diluted 4ml of blood sample using pipette (do it slowly to avoid mixing of the blood with ficoll due to force given during addition). Centrifuge this at 1400rpm for 30 minutes at 20 degree C (brake off)

After centrifugation, you can find buffy coat containing PBMCs over the Ficoll layer, as shown in the figure below: (hisep is commercial name of Ficoll)

Carefully the plasma layer must be discarded and the buffy coat is collected in a separate clean falcon (must be done with care to avoid mixing of layers). The separation of the cells is based on their density, Platelets are the least dense cells and they top the layers, followed by PBMCs which are also not dense enough to cross the Ficoll whereas RBCs and other WBCs are dense enough to cross the Ficoll.

The separated buffy coat is further washed with suitable medium at 1400rpm, 15minutes, 20 degree C (brake on). Again resuspend the pellet in appropriate medium and do Trypan Blue viability check assay and count the viable cells and find the number of viable cells.

And, normally the yield will be 0.5to 3 *10^6 cells/ ml.

Isolation of monocytes using Percoll gradient

Prepare Percoll gradient (using Standard Isotonic Percoll 9parts Percoll + 1 part 10X PBS) by using 0.15M NaCl. Generally, 60% (lower layer), 45% (middle layer) and 35% (top layer) percoll solutions are prepared for preparing the gradient. For a concentration of 3*10^7 cells lower and top layer of 2.5ml and middle layer of 5ml can be used. This is prepared by layering successive layers in a falcon, if required, centrifuged.

Then overlay the gradient with PBMC and after centrifugation at 1400 rpm for 30 minutes, monocytes can be obtained from the middle layer.

Hope, this helped you. any mistakes? Doubts? kindly comment!

Lowry Assay Principle and procedure

2013-10-24T08:41:00.001-07:00

Though there are several protein assays available, the most preferred one in many laboratories is "Lowry assay". It is effective in the concentration range of 0.01 mg/ml to 1 mg/ml. And, as an additional info, the paper published describing the procedure and principle of Lowry Assay is the most cited paper in the scientific history. (Feeling like, "Wow! I want to publish one to compete with Oliver.H. Lowry"??? :P)

Why Lowry?

Though there are several other protein assays, mostly Lowry assay is used in many laboratories. The reasons for preferring Lowry are: sensitivity of the assay, highly reproducible, cost effective, easy to perform. Biuret assay is generally used for higher protein concentrations like tissue samples but, Lowry for less concentrated samples and hence used in most of the molecular biology laboratories where there will be need for assaying comparatively less concentrated protein samples (in most cases where we attempt to produce enzymes).
Other assays like Bicinchoninic Acid and Dye binding assays are used in only specific circumstances and mostly not preferred due to its sensitiveness to contaminants like carbohydrates, lipids, etc.,

Principle behind Lowry's Assay for protein with procedure

The reactions that occur in Lowry assay are binding of Copper to the Nitrogen in the peptide. And, the phosphomolybdic tungstic acid in the Folin Ciocalteau reagent gets reduced to hetero poly-molybdenum blue by the copper catalyzed oxidation of aromatic amino acids in the peptide, in alkaline conditions. The assay must be done at the pH of 10 to 10.5 as it is sensitive to pH changes.

Reagents Required:

A) 2% of sodium Carbonate (50 ml) + 0.1 N NaOH solution (50 ml)

B) 10 ml of 1.56 CuSo4 + 10 ml of 2.37% Sodium potassium tartarate

Lowry's Reagent = 2 ml of (B) + 100 ml of (A)

Folin's Reagent what we use in our laboratory is ready made one (2 N) which is just diluted and made as 1 N for use. (by mixing with equal volume of water)

For constructing Standard curve, BSA is generally used. Stock of 1 mg/ml is required.

Note: Prepare all the reagents in distilled water. And, prepare the reagents freshly before use, just before use, to avoid precipitation of the salt added, also mix the reagents A and B only before use.

Procedure:

First the BSA stock is diluted for standard curve construction. Let the total volume be 1 ml, so for preparing a concentration of 0.05 ml, take 0.05 ml of 1 mg/ml BSA stock and mix with 0.95 ml of distilled water.
Similarly, we can prepare various standard concentrations like 0.1,0.2,0.4,0.6,0.8,1 mg/ml for total volume of 1 ml by mixing 0.1, 0.2,0.4,0.6,0.8,1 ml of stock BSA with 0.9,0.8,0.6,0.4,0.2,0 ml of distilled water respectively.
From this prepared standard concentrations of BSA, 0.2 ml must be taken for assay. For example, from the 1 ml of 0.05 mg/ml BSA prepared 0.2 ml must be taken in a separate test tube. Similarly, 0.2 ml must be taken from all the other standards.
Then, 2 ml of Lowry's reagent must be added to each of this 0.2 ml sample and incubated for 10 minutes.
Then, 0.2 ml of Folin's reagent is added to each of the incubated tubes and incubated for 30 minutes.
After 30 minutes, the tubes will have blue colored solution, which is further read at 660 nm in Spectrophotometer.
Graph should be plotted with absorbance in y axis and concentration in X axis.

And, using this standard graph, we can determine the concentration of unknown sample by extrapolation.

This is a very easy assay which I learnt in the very beginning of my course, but, still a very useful one and I'll be using it even after years I believe.

You know, I feel the major disadvantage of this Lowry assay is that you need to spend at least two hours to complete it including reagent preparation and incubation. (Sometimes, I feel like, oh, no, incubation for 30 minutes!How good it would be if there is no need for incubation?) And, when you don't have that much time, what will you do for assaying your protein?

It's simple, just measure absorbance at 260 nm and also 280 nm. Using the formula,

Protein (mg/ml) = 1.55 *(Absorbance at 280 nm) - 0.76 * (Absorbance at 260 nm).

This can be used when you have a spec which operates in UV range. No need for doing Lowry Assay, Yippee :P

Hope, this helped you :) Found any mistakes? Doubts? Let me know with your comments!

Cre lox system - Basics

2013-10-02T06:15:00.000-07:00

It's been a long time, since I wrote a post here. Finally, final year of my course and so very busy :) with books! And now, got time to share with you.

As you know already (don't know?, then get to know :P ), I use to have favorite subjects in each semester, third semester it was microbiology, then in fourth semester, it was cell biology, fifth was molecular biology, sixth was genetic engineering and now i'm in seventh semester, but, you know what, I don't have a favorite subject!!! Don't worry that I lost interest, instead, I got 3 favorite subjects - Immunology, Animal biotechnology and Plant Biotechnology!

It's awesome this time to have more than one favorite subject and here I'm gonna tell you something about, cre-lox system which is most widely used for making modifications (mostly deletions). These modifications can be done at a specific tissue alone by using a tissue specific promoter i.e., you can selectively knock out a particular gene in particular cells (for eg: hepatocytes) alone.

Cre recombinase is a protein which could bind with specific sequences called loxP sequences. To explain it in a simple way, the cre recombinase binds at loxP sites on either side of your gene (which is to be deleted) and makes a cut! This leads to removal of the gene and further the break is repaired using the DNA ligase of host system.

A single loxP site will have 13bp on either side of middle 8bp. The cre recombinase protein binds with this 13bp sequences forming dimer i.e., one loxP site contributes for a dimer (containing 2 cre recombinase units). Similarly, loxP on the other side of the target gene will contribute for a dimer formation. Then these two dimers will form tetramer (by folding the DNA strand as shown in figure. Then, cutting at the tetramer site happens leading to either translocation, inversion or deletion depending on the direction of the loxP site (i.e whether it is direct or invert repeat on either side of the target gene or located on different chromosomes)

13bp 8bp 13bp
ATAACTTCGTATA -NNNTANNN-TATACGAAGTTAT

This insertion, deletion and translocation could be done specifically at a particulat type of cell by using specific promoters. For example: To delete a particular gene in kidney cells alone, one could use kidney specific promoter for controlling the production of cre recombinase, so that cre recombinase will be expressed only in kidney cells. The molecular mechanisms of this deletions and recombinations invloves holliday junction formation. But, to be frank, i'm not clear or good at this holliday junction.

I''m trying to understand the molecular mechanism behind this, and, once I'm clear with it, I'll share it with you.

Understood the cre-loxp system? Got any doubt? anything wrong in my explanation? Kindly comment and I would try to reply or correct as soon as possible.

Diagnose your disease!

2013-08-13T09:22:00.000-07:00

Let it be any disease, ranging from a fever (which is the symptom of many diseases) to HIV, diagnosis at an early stage is very important for giving specific treatment.Whenever I go to a doctor, he asks me things like, stomach pain, fever, body pain, head ache, running nose, throat pain etc., then, he checks pulse, then he uses his stethoscope, he then gives all the possible antibiotics! Yes, really, this happens in most cases. Then, if my problem is not fixing up in a week, he'll ask me to take a blood test, this, that, and, all!

Nothing wrong in this, because he can't ask everyone coming to him to take blood test at the very first sitting, then, no one will visit him back saying he sucks out blood all the time! But, it is okay to have a blood test after a week, but, not after a month! Diagnosing a disease after it had reached a severe stage is comparatively less significant than diagnosing at an early stage. So, how to diagnose?

Antigen - Antibody!

Antigen and Antibody interactions are mainly used for diagnosing most of the diseases. When you give your blood sample to your doctor, he gives it to his lab, there they check for the presence of particular antigen in your blood. For example, if you have typhoid, surface proteins of the causative organism (Salmonella typhi) will act as antigen and when antibody (specific protein produced by your body against the antigen) is added to the infected blood sample antibody - antigen interaction occurs, which could be detected by various techniques.

Possible methods for diagnosing a disease!

There are several methods available for diagnosing a disease, but, most of them are based on the antigen-antibody interaction. Here I had listed a few methods of diagnosis, which I know:

1) ELISA - Enzyme Linked Immunosorbent Assay!

This method is based on the antigen antibody interaction. Two antibodies are used for detecting the presence of a single antigen. But, both the antibodies won't be directly reacting with the antigen, one antibody (primary) will be reacting with the antigen, and other antibody(secondary -which was raised against the primary antibody) will react further with the primary antibody used, producing color based on the enzyme linked with it. Generally done using 96 wells plate.

2) Western Blot!

This is also done for finding the presence of a specific protein (in diagnosis, the protein is antigen) in a given sample.This is done generally after doing SDS PAGE and transferring the protein separated in the gel to nitrocellulose membrane followed by similar technique of adding primary and secondary antibody, which will show color if there is infection.

3) PCR - Polymerase Chain Reaction

Though the presence of a particular gene could be detected using Southern blot, the most sensitive technique used for diagnosing a disease based on genes is PCR. Quantitative PCR could be done for even finding the severity of the disease based on the concentration of a particular gene of the causative organism.

PCR

I prefer to go with PCR than any other technique, but, when there is no thermo cycler in your laboratory it's better to go with ELISA! There are also other techniques like latex agglutination and microscopic observation of samples, but, the best way is PCR!

What do you say? PCR?

Possible mistakes while doing SDS PAGE!

2013-07-31T09:43:00.002-07:00

Hi, dear readers, friends, it's been a long time, I wrote here. Sorry for that, was a bit busy in lab! And, you can be happy because of the fact that I was busy, as I got lots of experience to share with you! Let us first start with SDS PAGE!

SDS PAGE - Sodium Dodecyl sulphate - Poly Acrylamide Gel Electrophoresis! This SDS PAGE is done for separating proteins based on their molecular weight. It is a widely used technique and it is very useful for having an idea about the expression of your protein of interest.

Principle:
The name SDS PAGE comes from the fact that this method uses SDS for making your protein uniformly negatively charged and of course, the gel is prepared using poly acrylamide. Why to make the protein negatively charged? Because, here our interest is to separate proteins based only on their molecular weight, but not based on charge and all proteins are not negatively charged like DNA (which is separated based on size using Agarose Gel electrophoresis). SDS is an anionic detergent and it makes your protein negatively charged. Hence your protein moves towards the positive electrode. i.e. anode.

SDS PAGE

Okay, the principle is fine, but, what are all the possible mistakes that one would do, while doing SDS PAGE? Here, I'll explain some possible mistakes, so that it would help beginners. I recommend you to learn the procedure for SDS PAGE before reading this.

While casting Gel!

The first possible mistake you would do is, regarding the volume of gel that you should prepare! The plates - thick and thin plates. The thick plate comes in different thickness like 1mm, 1.5 mm etc., If you are preparing gel using 1.5mm you need different volume of gel and different volume for 1 mm. So, before preparing the gel, be clear about the volume that you need. If you prepare more, then obviously, its wastage of chemicals, if you prepare less, it is waste of chemicals even now, as your gel won't come up to the top of the plate to dip the comb in (then you must discard and prepare the right volume once again)
Place the plates right in the gel cast right, check that they are equal i.e not up and down. If you place one slightly up and fix it in the gel cast, then your gel will leak out!
Check for leakage always with water before filling your gel in! Don't be over confident that your plates would never leak!

While loading your sample!

Check you are loading your sample only inside the well and not into the tank, because there are possibilities for mis-loading, so double check before releasing your valuable protein sample! (it's valuable isn't it?)
Remember to note down the order in which you load, because there is no point in running gel with out knowing the order of your samples in the gel! This might appear silly, but, happens!

While running!

keep watching your gel, else all your sample would run out! oops, waste of time, chemicals and your hard work!

Wondering? how I'm listing lots of mistakes? Because, I had committed some of these mistakes and I had seen people commiting the rest! Making mistakes, is nothing wrong, but, repeating the same mistake is!

So, remember these things, save your time and chemicals!

Oral Cancer & HPV

2013-06-20T22:38:00.000-07:00

Oral cancer refers to cancer in any part of your mouth like tongue, lips, palate etc., Cancer may be due to various reasons like irritation due to continuous alcohol consumption, smoking, poor oral hygiene etc., And it can also happen due to Human Papilloma Virus (HPV).

Human Papilloma Virus is a type of non enveloped virus (Papilloma) that can infect humans. They are classified generally as "High Risk" and "Low Risk" types. The high risk type HPV 16 is found to cause oro-pharyngeal cancer.

Diagnosis: Generally biopsy is done where a small part of your tumor is removed and observed under microscope. The biopsy can be just cutting a small portion or it can also be done using brush for collecting the cells.

After confirming the abnormality using this microscopic observation, further analysis is done for finding out the type of HPV which caused the oral cancer or to find whether the cancer is just because of alcohol/smoking.

This further analysis can be done by genotyping for exactly finding out the type of HPV. This genotyping could be done by several methods. Some of the methods of genotyping are

Line Probe Assay
Assay based on Melting Temperature
Using micro-arrays
Hybrid Capture Test
Multiplex PCR

All these methods have their own advantages and disadvantages. Among them, the multiplex PCR is found to be very effective.

Symptoms: Any visible tumors or lesions which remains uncured more than two or three weeks. Difficulty in chewing, swallowing etc., This might also be the symptoms of other problems, but, it is better to go and consult a doctor when you have these symptoms.

To avoid oral cancer, avoid alcohol, smoking and maintain good oral hygiene.

First few days at RGCB as SRF!

2013-06-10T12:11:00.002-07:00

It was 24th December I believe! I just saw the details of the Indian Academy of Fellowship only a week before the last date. I decided to apply for it and just applied in a hurry actually, yes, it was really a hurry! I was done with everything and sent the application.

I was waiting for the release of selected list of candidates from the first of March. They updated the selection list everyday but my name was not on the list till 25th of March! I used to go to the IAS website everyday and check the list, but, my name was not there in the selected list of candidates. I lost all my hope and decided that I won't get selected. But, it happened! I received a mail from the Academy after 25th of March! I was happy to the core, jumping up and down. Because, I was never out of my town from my child hood all alone. And, this fellowship was something really precious for me!

I believed that this would change my destiny to a better place! I was happy till my father said "no" to this. He was really afraid to send me out of my town! Yes, people in our town find it difficult to end their daughters for work or studies. But, I managed to convince my dad and he accepted to allow me to take up my fellowship.

And, the next problem after convincing my dad was, "when to join for the fellowship?" After lots of confusions, finally joined at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram which was allotted as a centre for my fellowship on 4th June.

And, this is the first time for me, away from home! That too, in another state where I don't know the language. I got accommodation in the Centre's hostel. My room mate, she is a Dehradun and I'm really enjoying her company.

IAS

In the centre, I got placed in Cancer Research Department and now I'm working on Tongue cancer. Cancer Research became my dream when I gained interest in molecular biology. And now, my dream is getting true!

You'll feel great when your dream is coming true and I'm feeling it now, right now! For many people this might sound a bit exaggerated, but, really only I can feel how much this is important for me! And, really I can feel the value of this opportunity.

This research experience will surely help me to get into a University for my graduation. And to the people who want to join RGCB, this is an awesome place, you can very well join here for better research experience! I recommend RGCB for getting Summer training.

In RGCB they provide accommodation for the fellows inside the campus in their hostel which is useful for the students from other states/ places.

The normal lab timing is from 9.30 a.m to 5.30 p.m. And students do research even late nights which shows their ultimate interest. People here are dedicated!

Learning many things apart from the academics, which will be very useful for the rest of my life. It is very safe in here and the place is really "God's own Country". People here are very friendly, smiling and caring, so that getting acquainted with the new environment becomes very easy!

Finally, I'm feeling free and independent for the first time in my life with a complete satisfaction of what I'm doing right now!

Want the IAS fellowship? Read "this" and apply next year! My Best wishes!

[ I did write this post as it might motivate someone or can serve as a piece of information who want to know about the SRF experience at RGCB]

Genotyping, Phenotyping, Karyotyping!

2013-06-10T04:37:00.000-07:00

We are going to discuss here about three different typing! They are genotyping, phenotyping and karyotyping. Before learning about them let us learn what is genotype, phenotype and karyotype!

Genotype, phenotype and karyotype

Genotype refers to the genetic make up of an organism. Generally the genes of an organism. The genotype of an organism can be represented as BB or Bb or bb based on the gene. If a person is having two recessive CFTR genes, then he will be getting cytic fibrosis. Genotype of an organism has also effect over the phenotype. Thus, genotype is representing the alleles of a gene in general. Genotyping is generally done based on PCR or hybridization.

Phenotype refers to the visible characters like structure, color and also the biochemical characters. This is based on the phenotype. Phenotyping can be done using biochemical assays.

Karyotype refers to the number of structure of chromosomes in an organism. karyotyping is done by staining and visualising the cells under microscope.

I'm in love!

2013-05-31T06:40:00.001-07:00

As a biotechnology student whether I'm taking care of my meals plate or not, I have to take good care of my petriplates. I wash my petriplates better than my meals plate!

Yes, we never autoclave and sterilise our meals plate, but we do for our petriplates! We ( I mean me and my friends) enjoy cleaning and autoclaving our petriplates in our department media room. And you know, my friends always complaint that I'm never accompanying them in cleaning. Yes, I'll go, wrap my petriplates, put it in autoclave, but I find it very hard to wash!

Always, I do autoclave, but, never wash after the autoclaving, I'll give that easy job to my friends :P I love my petriplates! Yes, I love them a lot.

But, how do I say that I love my petriplates? I love them because I'm always with them. I love them because, even whether I do complex Genetic engineering or simple Microbiology, I always need them!

And the final reason for loving my petriplates is they are so cute, especially, the glass ones! Wow, they are so cute!

And, today I loved them even more looking at the google doodle! Wow, Awesome it was.
For the 161st birthday of Julius Richard Petri - German microbiologist who invented petriplates, google had done this cool doodle. The six plates, each showing the source of the microbe when you place the mouse over it, looked really cool.

And, the best thing I loved was, Wikipedia has updated the information in the page of Julius Richard Petri adding, "google made a doodle for his 161st birthday!"

How fast, I had never noticed previously about this update of Wikipedia on google doodle, just today I noticed it and I was surprised! This is really really awesome. You didn't look at the doodle? Go, check it out, it is "sooper" :)

Love you Petri! <3 :) :P :)

Types of plasmid!

2013-05-25T09:59:00.001-07:00

I learnt genetic engineering, and of course, about plasmids. But, when I was questioned about the types of plasmid, God, I was blinking! :(

I had known plasmid only as a vector most times, I was aware of the types of vectors but, types of plasmids? :(

Okay, It's not a great shame, I learnt it now! Let me share here about the types of plasmids, just what I learnt.

Based on the functions of a plasmid they are classified as,

Resistance Plasmids:
These are the plasmids which have resistance genes. For example the gene coding beta lactamase which offers resistance against beta lactum antibodies like ampicillin. This is an important thing which is used while performing cloning experiments for checking the transformation. This was called as "R Factor" before the discovery of plasmids.

Fertility plasmids (F Plasmids):
And, these type of plasmids they have important genes called tra genes, never wonder, tra genes are transfer genes which are essential for the non sexual transfer in bacteria. This helps in formation of Pili and conjugation as a result.

Col plasmids:
These plasmids are the ones which have special genes for inhibiting the growth of similar bacterial strains i.e., bacteriocins.

Degenerative plasmids:
These are things which contains special genes responsible for digesting toulene like things. For example, I could say "Super bugs" they have engineered plasmid containing genes for digesting several types of chemicals and it is used for clearing oilspills.

Virulence plasmids:
These are the ones which makes us to hate bacteria. Yes, these make the bacteria virulent i.e., disease causing. You hate it, right?

Still I have one more thing to explain, "Promiscuous plasmid". This was asked in my university exam and adding to the sadder part, I didn't know the answer. But, nothing to worry, I got the answer now.

Promiscuous plasmids: These are plasmids which are self transmissible to a wide variety of organisms. They allow transfer of DNA to other species.

You know any other types of plasmids? Then share with me in the comment section, anything was not clear? comment then.

Take care. Bye :) :)

Be ready to fail, if you wanna be a Bio-technologist!

2013-04-11T06:37:00.000-07:00

Fail??? "Oh, come on, you should not say like this", is this what you are feeling, after looking at this title? If so, don't worry, I'm not meaning this in a negative way as you think.

"Everything in life has ups and downs. What about this field of Biotechnology? It's not preferred by many students. Yes, I agree, it is not like the IT field, you can't find a perfect job after completing just your under-graduation, even if you find a job, that won't pay you much. So, obviously, people will go for areas which would give them a bagful of money.

"You think, Biotechnology is worthless then? you say, you won't get paid?", is this your next question?
If so, I'm not saying that.

Biotechnology, here, you can achieve heights only after years of enthusiasm and work in your laboratory. It's not the matter of earning money. This field is for people who want to achieve or discover something which is in your own body, in plants around you in animals and in all living things around you.

And, now, we came away from our talk on failure. Why I mentioned like, be ready to fail. It's because, you won't succeed in most of your experiments in a single attempt! And, that's why I asked you to be ready.

When we try to clone in our lab, we repeated the ligation step twice, we tried different vectors even. And at last we succeeded, it's not like debugging a C or C++ coding where you could look into the coding with your naked eye, you have to trouble shoot something which you can't see with your naked eyes! Looks difficult? But, it won't be, when you love this beautiful field - Biotechnology.

Failures will become a part of your life and you will enjoy your success which comes after every failure. And, the moment see your experiment giving positive result, ah, that is happiness, the real happiness. Only you would feel this, if you were a biology person! (or a person who has maximum interest in any field.

Every little thing you do in your lab will fail, but, don't worry, it'll be followed by a success!

Southern Blotting / Hybridisation!

2013-03-31T11:14:00.000-07:00

Southern blotting is used for finding specific DNA sequence from a DNA sample by using the combination of techniques like gel electrophoresis, capillary electrophoresis and hybridization. The DNA from the gel (after gel electrophoresis)is transferred to a nitrocellulose membrane and hybridization analysis is done for finding out the sequence of our interest using a probe)

Let us discuss here the steps involved in doing Southern blotting. The steps are
1) Preparation of Sample
2) Restriction of the Sample
3) Gel Electrophoresis
4) Pre-treatment of the gel
5) Blotting
6) Hybridization

1) Preparation of Sample: (plant/animal/bacterial)

For tissue or blood samples the DNA could be obtained by treating with SDS (Sodium dodecyl Sulphate)
If we are going to have the DNA from a bacterial sample, there are separate protocols for extracting genomic DNA and pDNA. For extracting DNA from plants, CTAB (cetyltrimethyl ammonium bromide)
is used. This CTAB binds with the DNA and helps in better extraction using phenol. This CTAB is used specifically for plants, because they contain more carbohydrates than animal and bacterial cells.

2) Restriction of the Sample:

Specific restriction enzymes could be used fro restricting the sample of DNA. Only one restriction enzyme can be used or more than one enzyme could also be used. For restricting the sample of DNA containing 1 micro gram per microlitrethe following amount of buffer, enzyme could be used. (Choose the buffer corresponding to the enzyme you use) This volume could also be scaled up.

Components	Volume (micro litres)
10X buffer	1.0
DNA	1
Water	7.5
Enzyme	0.5
Total	10

3) Gel Electrophoresis:
Agarose gel electrophoresis is the next step. The restricted sample is run in the agarose gel (% of the gel depends on the sample)

4) Pre-treatment of the gel:
The gel is pre-treated with 0.25 mol/litre HCl for 30 min and also treated with an alkali.
The reasons for pre-treatment:

Acid treatment helps in breaking the DNA in the bands formed in the gel into smaller fragments and this also leads to little depurination!
Alkali treatment is done for denaturing the DNA by breaking the "H" bonds. This helps the DNA in binding easily to the membrane during the blotting. And also this helps in the hybridization with the probe.

5) Capillary Blotting:

This could be done by using nitrocellulose membrane or nylon membrane.

i) Using nitrocellulose membrane (high salt transfer)

While using nitrocellulose membrane, gel must be neutralized after the alkali treatment because at the pH above 9, there won’t be efficient binding of DNA with the nitrocellulose membrane. The neutralization is done by soaking the gel in Tris – salt buffer.

Blotting is generally done using nitrocellulose membrane. The capillary electrophoresis is set up as shown in the figure using SSC buffer (20X) (SSC contains 3mol/litre Sodium chloride and 0.3 mol/litre Sodium citrate) blotting for about 18 hours.

After the blotting or transfer is over, the membrane is washed with SSC (2X) and then dried. In this case, the DNA won’t be firmly attached to the membrane, so, the membrane could be baked at 80degree C for 2 hours which will result in semi permanent attachment of the DNA to the membrane

This same procedure followed for the uncharged nylon membrane also, till the washing and drying. After that for strong binding of DNA with the membrane, the nylon membrane is UV treated.

ii) Using a positive charged nylon membrane:

In this method, a different buffer is used and no alkali pre treatment of the gel is needed. Here, O.4 mol/litre NaOH is used as buffer and this gives immediate covalent binding of the DNA to the membrane and hence no baking or UV treatment is needed.

Advantages of nylon membrane :

i) Better binding of DNA, even 50bp DNA binds efficiently whereas nitrocellulose membrane efficiently binds 500bp only.

ii) Nylon membranes cannot be damaged easily and hybridization could be done repeatedly upto 8 or 10 times.

iii) No need for post blotting baking or UV treatment for +ve charged nylon membranes and has reduced loss of DNA.

Electrophoresis or electroblotting from the gel to membrane could be done instead of capillary blotting and also vacuum blotting which draws the buffer through the gel to the membrane could also be done. This reduces the time of blotting to a few hours to minutes.

6)Hybridization:

Pre-hybridization step: This is done to block the areas of the membrane which donot contain DNA, to avoid random hybridization during hybridization. Generally this is done by treating the membrane in the buffer containing Salmon sperm DNA (popular choice of DNA used for blocking) for 15 minutes to 3hours depending on the membrane type.

Then actual hybridization is done by using high salt buffer with a detergent (2X SSC + 1% SDS) containing the probe DNA sequence. For increased sensitivity 10%Dextran sulphate or 8% polyethylene glycol 6000 is added to the buffer and probe mixture, this increases the cross linking between the probe and sample DNA. For increasing the specificity, the temperature at which the bonding between the probe and the sample DNA will be stable must be used; at this temperature all the other random bonding will be unstable.

Thus, we could identify the DNA fragment containing our interested sequence using Southern blotting.

Any queries? feel free to comment.

Ref: 1) Southern Blotting and Related DNA Detection Techniques

By, Terence A Brown,University of Manchester Institute of Science and Technology, Manchester, UK

2) http://www.nature.com/scitable/content/southern-blotting-and-hybridization-with-probes-can-44927

Plant Bio Reactors (PBR)

2013-03-19T07:48:00.000-07:00

You might be aware of using E.coli for producing human proteins. E.coli is a well established expression system and easy to handle. And many people are working on this E.coli (even I'm handling only E.coli in my laboratory most times)

Okay, we are fine with E.coli, and we are producing our protein of interest in that. And what is the need for an eukaryotic expression system? That thing, I explained in one of my previous posts (Clone human genes into plants!)

I explained in that post like we are expressing our human proteins and we are growing the entire plant for obtaining our protein of interest. And we need not do that all time and there is a choice of growing plant cells in bio reactors!

Yes, we do grow E.coli in bio reactors and why not the plant cells? You might be aware of this plant bio reactors and here I'm going to share something that I know about them.

Plant bio reactors

I attended a seminar on "Advances in plant biotechnology" and as you guessed, this post is the result of that.
Okay, let me jump into the topic.

Important factors to be considered while designing a PBR are:

Plant cells (as well as animal cells) are shear sensitive
Plant cells form aggregates - and this aggregate formation sometimes helps you with your production
Risk of contamination

I consider that these three things are very important in growing your plant cells in a PBR.

Shear sensitivity:
As, the plant cells are shear sensitive, we can't use the same reactor which we use for growing our microbes for growing plant cells. If you use a super fast impeller for effective mixing to eliminate the mass transfer limitations, then I'm sure your plant cells are gonna die. So, special design is needed for growing the plant cells.

So, there must be a balanced mixing such that the mass transfer is good and also the cells should
not lyse!

Aggregate formation:
Plant cells also form aggregates like bacterial cells. We don't prefer aggregate formation in case of bacterial cells. But, in plant cells, depending on the species, this aggregate formation could improve our productivity.
On the other hand, this aggregate formation causes mass transfer limitations thus affecting your productivity.

"???" You feel like "oh God"? And your question is "whether to have aggregates in plant cell reactors or not?"

Let me answer you. You need to have aggregates if it benefits you with your productivity but the same time, the mass transfer must also be good. For having the two things in balance, we have to maintain a "particular aggregate size", so that, our productivity will be fine in both the aspects like mass transfer is good and aggregate formation improving the productivity.

Then, the next question may be "how to maintain the optimum aggregate size?"

The optimum aggregate size could be maintained by using immobilization technique. You could immobilize your cells i.e. the optimum aggregate size would be maintained using immobilized plant cells.

Risk of contamination:
Comparing the bacterial cells, plant cells have a very high risk of contamination. Even when we grow them for producing callus, there is a lot of contamination risk especially fungal infection.

So, growing them in a reactor, that will be difficult job especially in case of maintaining the sterile environment. Most times, continuous reactors are not used for bacterial cultures as it has high risk of contamination comparing the batch and fed batch. I had never seen or done continuous culture of bacteria in our lab, normally we don't do continuous culture as it goes for more than a day and also it is difficult to maintain sterility. And, even when on of my seniors wanted to try continuous culturing, we were all saying "no you better try batch instead" fearing that everything may get spoiled due to contamination.

But, if high sterility is maintained, you can do continuous culturing of plant cells also.

Hope you understood what i explained, got doubts? Comment or e-mail, will answer you as soon as possible.

Tuberculosis Diagnosis!

2013-03-03T07:31:00.000-08:00

Hi, Today I'm going to share something about TB! TB - Tuberculosis is generally caused by Mycobacterium tuberculosis. The most common symptom of TB is "non -stop" cough!

They say, many people in India are "carrier" of the bacteria, but not the disease! Oh God! if you are in India, better go and check for this bacteria! The possibility of the spread of the disease could be due to unhygienic conditions!

You spit in public places? you sneeze? cough? No problem, you can sneeze or cough, but be careful! Careful coughing? careful sneezing? yes, you might spread the population this mycobacterium you are carrying (you need not have TB), so, be careful in sneezing and coughing!

Okay, let us come to the point, I started typing this with a view of giving an overview about "the diagnosis of Tuberculosis".

The most common test used for checking TB is "Tuberculin Skin Test". Let me explain you how this is done!

Tuberculin Skin Test!
Let us first look into the principle behind this test. When a person is infected with Mycobacterium tuberculosis, then, he will be carrying antibodies against it. When he/she is injected with the antigen from this bacteria, he will produce an induration (hard bulge in the place where the antigen is injected). This could be found after sufficient time like 72 hours.This is used for diagnosing TB! In this case, false positive results are obtained when a person tested for the second time using this method.

Antigen? What is the antigen used here? Is that your question? It is the protein which triggers the immune response extracted from the mycobacterium sp.

Do you think this is enough for diagnosing TB? "Okay, what else one could do for diagnosing TB?" When my professor asked this question in class, I replied "ELISA".

ELISA - Enzyme Linked Immuno Sorbent Assay: Here the blood sample from the patient must be collected and checked for antigen antibody interaction in the well plate. Here we must have the antibody available in our lab. If there is antigen antibody interaction the ELISA produces colour else it won't!

"Okay fine. What else you could do for diagnosing TB?", when my professor questioned further, I replied, "PCR"! He was happy atlast that I had answered as he expected!

PCR: How PCR could be done for diagnosing TB? Mycobacterium genome sequence is available online in the databases and one could design primers based on that and when PCR works, then the person is having TB, else not! Real Time PCR is generally preferred than the normal PCR for avoiding errors.

The advantage of PCR is that one could also say the severity of the disease and dosage of the antibiotic could be given accordingly.

After all, the dosage must be very correct and the antibiotic treatment should not be dropped in the middle to avoid development "multi drug resistant" species!

I had shared whatever I understood as an overview, any doubts? mistakes? suggestions? Feel free to comment!

Clone human genes into plants! - Avidadham - International Conference on Molecular therapeutics!

2013-02-09T00:36:00.000-08:00

E.coli VS Plants!

As an undergraduate of Biotechnology, I know about cloning. I know about cloning a gene which codes for a human protein into a bacterial cell, E.coli, most times.

I also know about producing transgenic plant varieties. But, I don't know about cloning a human gene into plant! I came to know about cloning a human gene into plant and producing the protein in plants from the workshop session of AVIDADHAM'13.

Let me explain something about this!

As you may know, eukaryotic gene has both introns and exons. So cloning a eukaryotic gene into a prokaryote will have lots of problem like splicing and post translational modification.

Generally for cloning eukaryotic gene into a prokaryote like E.coli, we do follow bottom up approach where we use mRNA to synthesise cDNA, to remove the noncoding introns. But, even if we remove the introns there will be problems with post translational modification.

So, to avoid this, we are cloning our gene from human into plants? Here, we are not going to grow the plant cells, but, the whole plant.

Plant tissue culture

I had come to know that they had successfully expressed the protein of interest in plants but the problem is with purifying and extracting the protein from the plants. Once this is possible, once the extraction procedures are found, once the extraction is done with out affecting the protein's activity, then, I'm sure, this is going to be a ground breaking technique.

We could get bulk amount of our protein. But there are lots of problems when we come to the ethical side, these GM plants should be grown only inside a green house, so that it won't affect the other wild varieties by pollination.

If we are growing this in open field then due to pollination there is a possibility that our wild varieties might become extinct. So, we must grow these plants inside the green house.

After all, if we come out with an idea saying "GM plants", there are people who are ready to boycott!

But, I got very much excited about this idea of cloning human genes into plants. It's awesome, isn't it?

Any queries? Feel free to comment! I'll try to answer my best!

How to clone a GENE?

2013-01-21T18:12:00.000-08:00

Hi,

Wish you a very happy day! Let all you researches result only in success! J

You know, I’m learning cloning, wow, rhyming it is, learning – cloning!

Like this rhythm, cloning is also fun! Yes, fun, interesting! Let me share with you, what I know about cloning.

The first step of any cloning experiment would be selecting a vector. Now, I’m learning cloning using plasmid as a vector.

So, you have to choose a vector for cloning at first. Vector is nothing; it’s just a plasmid DNA into which we can incorporate our gene of interest and transform the vector into a host which will produce the protein coded by our gene of interest.

Vector need not be only p-DNA, viruses could also be used as vector. But, here, we are going to have an overview of cloning using Plasmid vector.

Okay, we had selected the vector, let us assume. What next? We have to look at our gene of interest now. We have to get the sequence of our gene of interest – it’s available in databases like NCBI.

Then, we have to isolate our gene of interest from the total genome of the organism. If it I prokaryote then you can just easily get the total genomic DNA and do a PCR (polymerase chain reaction) for amplifying your gene of interest.

If it is going to be an eukaryotic gene, then you must go in reverse. You must get the total RNA, then from that you have to isolate the mRNA, then from the mRNA you have to get your amplified gene of interest.

Before doing PCR, you must design Primer. The primer must have the restriction sequences at the end, so that the restriction sequence will also get inserted with your gene of interest at the ends.

Then after PCR, the gene f interest could be inserted into the vector just by restricting both your gene of interest and vector. They both will have complementary cohesive ends, so that they could hybridize with each other.

Of course, here there will be nick, but, we need not worry about the nick, once we transform this vector with gene of interest into our host – say E.Coli DH5 alpha, the host mechanism will take care of the nick.

E.coli DH5 alpha is a special strain of E.coli in which the defense mechanism against the foreign gene is knocked out, so that we could transform our gene into it and ask the E.coli to produce our protein, with out any problem of defense action.

This is just an overview of Cloning. There are several things to explain than this.

I’m happy that I know how to clone now!

Happy cloning!!!

Precise DNA cut!

2013-01-05T21:36:00.001-08:00

We all know that we can cut and engineer our DNA or DNA of any other organism. But, so far the methods used for DNA cutting are not precise.

Available techniques:

By the older method, the genes are added into the cell and they get inserted into the genome of the cell in a random manner. This can’t be used when our aim is to engineer the DNA at a specific site and here we can’t also replace the already existing gene with our gene.

There is homologous recombination method, but unfortunately natural recombination won’t occur in cells that easily!

There are also methods which use Zinc fingers for delivering nucleases which cuts the DNA, but, this is really hard, because nuclease can’t target every possible DNA sequence. This is costly too.

TALENs – Transcription Activator Like Effector Nucleases – cut the DNA at specific location. But, what to do, this is also costly!

Our new method

Here, we are going to use the already existing proteins of bacteria and RNA. The tool contains RNA sequence with the nuclease called cas9.

Here, RNA is used for recognising the site on the genome where we have to make a cut and Cas9 for cutting! The Cas9 get activated once the RNA finds exact match. It won’t get activated, even if there is a single residue difference.

If we are willing to replace the already existing sequence with new one, we have to provide a DNA template.

This is not costly, yet effective and precise!

This is a good news for all Genetic Engineers, right? Even, I’m happy about this as a GE student, then for sure this is going to be a boon for Genetic Engineers.

After all, as my professor said, Playing with genes is the real fun! J