Saturday, October 17, 2015

Care when you CLONE!

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!

Friday, June 26, 2015

The Butterfly flew for the first time!

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.  

Sunday, January 18, 2015


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 :