Whole Blood DNA Isolation Protocol

I am writting down the protocol for the DNA Isolation via Salting out process from blood. Gives good yield of DNA. A260/280= 1.6-1.8 (best)

Reagents

1) Solution A Haemolysis Solution * Prepare fresh or use a solution prepared in 24 hrs and stored at 4'C.

Final Volume (ml):          1000                    750                     500                  100

Sucrose (g):                   109.5                   82.125               54.75                10.95

1M MgCl2 (ml):                5.0                      3.75                   2.5                     0.5

Dissolve the above in autoclaved water. Do not autoclave after dissolution as it can char the sucrose.

Add Triton X @ 1% of the final volume.

2)  Solution B  WBC Lysis buffer * Prepare fresh or use a solution prepared in 24 hrs and stored at 4'C. Add SDS prior use.

Final Volume (ml):           100                      50

1M Tris-HCl;pH ~8 (ml):   40                        20

0.5M EDTA (ml):              12                        6

1M NaCl (ml):                   15                        7.5

Dissolve in dd.water. Autoclave and then add 20% SDS @ 5% to the final volume.

3) Solution C    Protein ppt. buffer * Light sensitive, so store in amber bottle at 4'C; storage can be done for 1 year at the max.

This solution is 5M Sodium perchlorate= 70.23g of sodium perchlorate in 100 dd. water/ autoclaved water.

4) 0.9% NaCl (mammalian saline)

5) for 200ml TE: 2ml 1M Tris/Trizma(Sigma) + 0.4ml 0.5M EDTA. pH: 8.4

6) Cloroform (in 4'C)

7) 70% Ethanol (in 4'C)

8) Isopropanol (in 4'C)

Procedure

S1. To 1 volume of heparin added blood add 5 volumes of 0.9% NaCl. Mix by inversion. Centrifuge at 2000rpm for 10 minutes.

S2. Decant the supernatant.

S3. Add solution A- 4 vols of the original blood taken and mix slowly by inversion. The solution will turn clear red due to lysis of RBC. Spin at 3500rpm for 5 minutes. If a white pellet is clean, proceed to next step or else any red pellet indicates incomplete RBC lysis. Add 2ml of Solution A and repeat the S3. Discard the supernatant.

S4. Add 2ml of Solution B, vortex to dislodge pellet. Invert mix for 10minutes unless the pellet looses it's integrity. (To hasten the process, I suggest to keep the mix in 37'C for 5 minutes in between two inversions of 7 minutes and 3 minutes.) If the lysis of WBC takes place, the pellet looks slightly thready and the solution gains a silky appearance of khadi silk.

S5. Add 500uL of solution C and mix rigorously using vortex.

S6. Add 2ml of chilled chloroform and mix. The solution turns milky.

S7. Centrifuge at 2500rpm for 5 minutes.

S8. Transfer the clear aqous layer in 1.5mL tubes. This may take several turns to transfer all the Aq. supernatant. The suggested process:

            S8/1: Take 500uL of supernatant and add 500uL of Chilled isopropanol, Invert mix by hand. Appearance of translucent threads. These are alcohol insoluble DNA.

            S8/2: Spin down at 13000rpm for 5 minutes or 10000rpm for 12 minutes.

            S8/3; Keep the pellet (DNA) and discard the supernatant. Repeat S8/1 unless all the aqous layer from S7 spin is accumulated in one 1.5mL tube for a particular sample.

S9. Add 1ml of chilled 70% ethanol. Wait for 5 minutes and spin at the speed conditions of S8/2.

Discard the alcohol and air dry. (a maximun dry of 18 hours is allowed; overnight dry). Keep in mind, not to over dry!

Add 100uL TE to the air dried pellet. Keep in 4'C for 3 days, before estimation and analysis.

Hope this will help a lot of my molecular biologists in different arena of life science. This is a tried and tested protocol.

Tell me how to win your heart...

For I haven't got a clue-

But its always you and me and everything new!!

Five years of walking together under the welkin, its approaching a new beginning. Its calling for the violin, the cakes, celebrations and many more. But when the time for these will run down, its just me... and you!

Changing defines my life, since when I met you. From the novice walker in the park, you made me transform into a steady feet! You have pulled me out of the several ideas of changing tracks. You derailed my corporate "passion" to the real me; making me comfortable in what I did and do and will keep on doing. May be that's the clue where I won your heart and you- won it long before you even thought about winning it.

Hello.......

Its just me and you.

Story of a Little Indian State: Himachal Pradesh (Part 4)

The fourth part of the story was long due.

It was time to move out of the State Capital and romp towards the northern Himachal, 300KMs from Shimla, to a famous tourist destination called Manali. (Not Manila, as many flips their tongue!)

Manali is beautiful, no doubt, but this post is based on the beauty that the roads towards Manali that holds the lens (eye/camera), pop out of the window; throughout the journey. Residing at the foothills of the Upper Shivaliks, near the meet point of Beas, Manali is like Haridwar for Beas. For more than 70% of the journey, which took a good 6 hours in the Alto, we traced River Beas. The pebbles of the river bed executed some of the colours, close to nature.

The river had its own succulent nature. It accelerates suddenly, while slows down the flat valley. On either side lies the barren apple orchards. sibce we visit just after the apple's season, we missed the fruit from heaven, hanging, just like a common "wild" fruit around! Our driver was caring enough to tell us the geographic nature of the place and advised us that Manali and Shimla has two well defined seasons. One must visit in both the seasons, to savor the taste of Himachal. Its April-September (Apple and Sunny time) and November - February (Snow time). I hope to visit on the later part soon.

The vitality of the trip was fresh air and soothing feel. On the way, we went through a newly made tunnel, that was dark, adventorous, four lane big, bypassing underneath the Himalayas. Its 3.1Km long, but thanks to the fading memory and age catching up, I just dont remember the name.

But, the most interesting and beautiful place that I saw in this whole drive of 6 hours was 20KM away from the Manali township. Inexplicable; that's why, I allow my pictures to talk for what I mean!

Lovely!

6 years, many new things to learn!

... and I just feel I have just started; although the feeling gets interrupted abruptly, as you can see the dropping stats in my frequency in blogging out thoughts. But, guess, that's how life moves into senescence drive from adolescence! Blog too follows the rule as its life, itself!

Since 2006, my romance with this small space had started and I have tried to make it large. My history claims that I have had 1,33,200 hits since 2008, which makes a good 26.6k hits per annum. Only that my adsense doesn't pay up much as my friends only love to read my thoughts rather than search some items or products from my blog! Come-on! i am not Amazon.com! Am I? (but sometimes money also makes me miss it's presence by "selling" my thoughts)

Some simple stats:

Till now I have had posted 450+ articles. Many have had received it's comments and due share of respect, with people liking it and mostly are being channeled by Google Search and many through facebook and previously through orkut and now a days, sometimes, Twitter! I have shared some 1600+ photoes, of which 982 photoes are my own copyrighted snaps! And I flaunt them to the world. And I am not finished yet! Infact, its a long way to go: Miles to walk, before I sleep.

copyright: Somnath Paul | 2012. Bagmundi, Puruliya | WB

But, today I shall be speaking a purpose. A purpose that makes a living. A purpose that creates a living and it makes you wonder, whether you are right with or not.

Back, last week-end, I was in Bagmundi, Puruliya. Having finished my work, I managed to move out into the sun with my lens to collect some memorablia from such a serene beauty; of raw nature. A small girl came to me. In bengali, she asked: Where did I come from? Why was I there? I told her that I went there from Kolkata and I work in IICB. (She looked awed; understandably, she didnot understand what I meant in the latter part.) She asked: Was I a reporter? The reason was obviously hanging from my neck. I smile a No! I asked her why she was asking me so?

I was just dumbstruck with her reply.

In bengali she said: I thought the reporters are here and that there would be trouble between  red and green and people from both colours would come into their village to torture them, followed by the men who has camera like mine. 

To an adult Bengali, the Red and Green meant to faces of the coin at the Bengal Bidhan Sabha. That small conversation of 5 minutes left me in wonder as to what happened to these people in the past two years; thanks to the tag: Mao-baadi or Maoists! 

All are same. But, then...

Life goes on!

Life for Others, Smile for Us.

Many movements have been organized all over the word, to bring and shape (also reshape) the human society, culture and civilization. I am happy, that I am part of one such movement, myself. Something that I loved doing and will keep on supporting till I am able to.

Rakshabandhan For Education (RFE), is an non-government organization, gathering funds to enlighten the the poor backward areas, with the light of knowledge, making them enlightened and getting a smile in  return; just to smile back. I, Somnath Paul, am a scholar from Indian Institute of Chemical Biology, Kolkata and am loving my activity as a member of RFE-IICB Chapter and our tryst to bring forth knowledge.

What is RFE?

An NGO that solely believes in making good talent shine through proper channels and for that the paper support (be it monetary or books) is provided by the organization.

What is RFE-IICB Chapter?

We, the people from IICB, believe in donating some of our allowances for the noble cause. We basically target in creating book-bank for the West Bengal Board syllabus in Class IX and X, in some of the needy schools in remote areas.

In the last few years from 2008; we have covered a school in Kultali, (interior of Sunderbans, Medinipur) and presently we are doing our work in two schools in Bagmundi (Purulia). Both places in West Bengal.

Since I went in the latter project, I hereby post some of the snapshots from Bagmundi.

All from Dhunuri High School, Bagmundi, Purulia.

The main purpose of this article is not to boast around the good work, but a request to all my scholar friends round the state, country and the globe. If you believe that the education that we received, ought to be shared, then  you contact RFE (click here). Any sort of help or participation is welcomed.

After all, Education is the Life for Others (under privileged), but Smile for us. 

So lets spread the Smile. 

Lets walk the mile; together!

Kony 2012: A Movement with a difference

Search google with the sun-storm of 2012, and every fifth link, you will find the movement impregnated, called Kony 2012. Its a movement, for a cause. Normally, with the modern political scenario like the Wall Street Fiasco later last year in USA or the Saddam Mission in Iraq, Gaddafi Movement in Libya or the Laden Assassination in Pakistan; a movement is mostly against the governmental efforts. Take for the Indian scenario. From Union Government's head-ache with Anna Hazare to the Stately matters of Mamata in West Bengal or Maya-Mulayam in UP, movements have shaped governmental policies from array to disarray or vice versa! In short, its against them.

But, movement Kony 2012, is to plead the government (international body) to take steps in arresting an atrocious dictator of Uganda,. Joseph Kony, who walks in the worng path of the righteousness, mutilating the verses of The Ten Commandments, to whip out gains from the Ugandians!  Kony 2012, is the motion to make this man famous, so that even the sleeping governments in Antratica can hear and take actions against this atrocious individual. He, who kidnap children to train them into terror commandos while make the girls item of sex and pleasure for the fighters for him. 

Its a bid to make him famous. I have done my part... read and share and do your part as well.

Kahaani: A Story of Anticlimax

Even the Gods admit: "Superb".

The movie will make money, but it will keep the legacy of being a thriller, where woman-hood wins the theme. I don't believe in writing down the script, as its upto you to pay and cherish the charisma, I just want to pen down the impeccable work by director, Sujay Ghosh and the acting by Vidya Balan and Parambrata. Revolving around the festive season of Durga Puja, the film upheld the tradition and rituals that the Bengali Festivity offer as it worships the strength of the Goddess. Just like Goddess Durga killed the demon (evil) Mahisasura, unvieling all the deceptions possible; the same is refelcted here.

Since, it is released on 9.3.12; hence, I would not kill the interest that the film deserves. You should go and watch it, as its better than Gupt. Dynamic, subtle and juicy! Just tailor made for near three hours of entertainment (including the commercials). You will love it!

I give it 9/10, not only for entertainment, but for the food for the brain to work! Its complete! Kahaani will Rock!

The Great Wall of India

This is what I wrote on Facebook Today:

 I used to hear from my dad and grand dads, about the pace of 60's and 70's, from the Windies. I will tell my offsprings and theirs', about the blades of 90's and 00's, from the Indies. One of those blades, retires today. The Wall, Mr. Rahul Dravid- You will be missed. You gave the country a lot; that cannot be defined. Thank you for standing in the middle!!

True, to the realization, when I was browsing the channels after a day's fight amongst colours and booze, I was throttled with the news, followed by several memorable matches and years that this man has given to the Indian as well as World Cricket fans.

Clubbed into the 10,000 runs getting players' Hall of Fame, Mr. Dravid have been  an inspiration to many hopeless, in several occasions, when he pushed the brakes to the India Wicket Slide. Also considered the member of the emminent Fav Four Tendulkar, Ganguly, Dravid and Laxman, spelled doom, any day.

The most mesmerizing part of this quadra, was that, inspite of the Master Blaster (Tendulkar); the other three played with their own natural flair and dominated every niche that the Wisden for Batting wanted. To personify them, compared to Marvel Comics superheroes, I would put down the following, that defined their powers:

1) Sachin Tendulkar:  He plays like me: Sir Donald Bradman. His powers lay within the name. His bat spoke every turns and indefinite corners of the spherical cricket field, devastating bowlers at will. s long as he was present in the field, Indians know that the match can be won.

2) Sourav Ganguly: On left side, there is first God and then there is Ganguly: Rahul Dravid. The Indians knew that with a fighter in the middle, this match will not go down the drain. The tantalizing square drives and the cork screw sixes off the spins, made Ganguly the most beautiful batsman to watch. He inspired several comebacks and still people say: He left the game too early!

3) V.V.S Laxman: Stylish: Geffrey Boycott. He controlled situations when India stared at a follow on! Poeple knew, we still have Laxman. Infact, it is the duo of Dravid and Laxman that stalled the victory chariot of Steve Waugh's Australia, from not setting the World Record of Test wins at a stretch to 16 only. It was Laxman and Dravid that set the pace, and Turbinator (Harbhajan Singh), turning the heads. In 2001, Australia lost to India even after India faced a Follow On, Eden Gardens, Calcutta (Kolkata).

4) Rahul Dravid: The Wall: Cricket Fraternity! And he justifies his name.

There are abstract words to describe Dravid. Just wishes, as the sun wakes up in another 5 hours, while another chapter of glorious Indian Cricket closes!

Fav- Four (l-r): Sachin, Sourav, Rahul, Laxman

SSRL

Courtesy: http://cricages.com/wp-content/uploads/Fab-4.jpg

What to write?

It seems that with dried up funds, my blog posts have also dried up. Rather say, I spend so much time in brain draining, on my research, that I find subdued times to work on this aspect of my life: Blogging! Never the less, I am on the blank canvas and am ready to paint it with what have been in and out in my life for the past month.

Not a digital diary; at least I shall try not to make it one.

With Sandip tying the knot with Susmita, Debmita taking her step into the life of being a man's woman; life have been eventful. And then, there is always the hard fact- research and PhD! Sometimes, its easy to find your way in the dark than finding your way up from the shallow well! I'm just having the latter part of the preceding statement. When, you talk about how you are; I just say- cholche, which if converted from Bengali to English, means- going along! But how? Not that is the question my dear.
Q1. Running? Nope!
Q2. Walking? Sometimes!
Q3. Limping? Definitely!

And then I come across hurdles. They kill you, but thanks to my inner spirit I make it smoothly, covering the sweat with a smile. To define with some tune it, you should definitely listen to this Bolly Song, that I came just the other day, while watching the movie: My Friend Pinto. Dhinchak Zindegi!

Its life, and all about it!

So, here to the celebration of ties being managed, over the past 29 days:

Debmita Weds Anirban

The Gang Debmita's Marraige

Sandip Weds Sushmita

S5 at Sandip's Marraige

Now, thats it.... Nothing more to write.