Saturday, July 28, 2012

Electroporation (Transformation)

Wish you baskets of smile :D :)

Last time, I explained about,  Electrocompetent Cells of DH5 alpha. As a continuation, this time you are gonna know about, transforming those electrocompetent cells with Plasmids. The transformation technique is very simple.

Last time I didn't explain you about the procedure to prepare electrocompetent cells. Let me give you a short  

Procedure to prepare electrocompetent cells:
1) Inoculate the E.coli DH5 alpha cells the previous day in LB broth.
2) Subculture it, the next day such that you have initial O.D (Optical Density) of 0.05
3) After two hours, you must have an O.D of 0.6 to 1
(This is because, the cells will be in log phase or in active metabolic state in  this O.D)
4) Then, ultra centrifuge your culture to separate the cells.
5) Ultra Centrifuge twice with cold water and one time with glycerol.
6000 rpm for 7 minutes at 4 degree c with cold water
4000 rpm for 5 minutes using glycerol.
6) Now, your electrocompetent cells are ready, put them in eppendorfs, store them in deep freezer with liquid nitrogen for further use ( - 80 degree C)

( I was very much exited when I saw the liquid nitrogen and deep freezer for the first time :)
That's why I always enjoy my lab hours)

The exact mechanism of altering the cell permeability by this procedure is not known.
The centrifugation with cold water is done for removing salts and ions present in the cell.
Glycerol acts as a cryoprotectant, maintains osmotic pressure.

Okay, lets go for transformation procedure now.

Things you need for this transformation are: 
Electroporation cuvettes, Electroporation system, Laminar air flow hood, incubator.
These are the little sophisticated things you need apart from, Pipettes, petriplates, culture tubes etc.,

1) Thaw your electrocompetent cells stored at -80 degree C using ice. 
2) For 20 micro liters of Cell, use 100 to 200 nanogram of plasmid DNA, mix and tap, leave in ice, for15 minutes.
3) Transfer this content from eppendorfs to electroporation cuvettes, and give pulse using electroporation system. ( During this electric shock, the DNA will enter into your electrocompetent cells)
4) Wash your cuvettes with LB broth and then tranfer the content to culture tubes else you can also use the cuvette  during the incubation. 
5) Use the Electro competent cells as your Control. (Give shock to these cells also, though they don't have  DNA to get transformed)
Incubate your cells for 1 hour under appropriate conditions before plating.
6) Plate your Cells and cells+DNA using LB ampicillin plates (Spread plate method)

Observe your plates the next day after incubation,
The cell + Plasmid DNA (with ampicillin resistant marker) plate will show growth, if transformation is complete.
The Cell alone plate won't have growth.

I asked you to incubate your cells before plating them. you know why??

Because your transformed cell won't be having enough plasmid to resist your ampicillin. So, if you give them an hour, their pDNA will replicate and produce enough copies to produce enough beta lactamase.

You need detailed procedure?
Then comment your mail i.d. 
I'l send you.

Any doubts, queries? mistakes? feel free to comment.

Sunday, July 22, 2012

Electrocompetent Cells of DH5 alpha

Electro competent cells are prepared for DNA transformation. Usually the cell wall of bacteria are very thick and hence, a foreign plasmid DNA can't penetrate into it, very easily. So, we prepare electrocompetent cells, which have reduced cell wall thickness or pores over the cell wall.

So, due to this pores, DNA can easily enter the cell and thus transformation is made easy. In our lab, we did prepare, electrocompetent cells of E.coli DH5 alpha. The procedure for preparing electrocompetent cells is available in various sites. So, am not going to discuss the procedure. If you wanna know it, kindly mail me, I will send you.

Okay, following the procedure, you can prepare electrocompetent cells, but, how to ensure that you had prepared a non contaminated one? Generally, Plating is done.

Just streaking, a LB plate and an Ampicillin LB plate with the cells. You must observe, growth in LB plate and no growth with the Ampicillin plate. If so, then pat your back, you did it.

What's the basic behind this plating? Why an Ampicillin plate?

Generally, E.coli DH5 alpha is not resistant against ampicillin. It will show resistance only after you transform it with a plasmid. (Plasmid gives resistance)

So, If you have contamination with other microbial cells, you will find growth in LB Ampicillin plate too.

:) Got it? Any queries? Mistakes? Comment then! :)

Happy learning! :)

Purine base pairs with purine & pyrimidine with pyrimidine!!!!?

I started learning molecular biology this semester! And, I'm very happy about it. As I love Cell and micro biology, I started loving molecular biology too. :)

My very first beginning with molecular biology is DNA. We all know very well about the structure of DNA as proposed by Watson, Crick, Rosalind Franklin & Maurice Wilkins. 

Certain basics about DNA:
1) DNA is Double stranded and acidic. They have four nitrogenous bases ATGC (Adenine, Thymine, Guanine, Cytosine), Deoxy Ribose, Phosphate. A&G are purines. C&G are pyrimidines.
2) The acidic charge is due to the phosphate group which is protruding out of the DNA.
3) A always basepairs with T & C with G. Between A&T there is a double bond of hydrogen. And, triple hydrogen bond between C and G.
i.e., Purine basepairs with pyrimidine or the viceversa.

But, why not a purine and purine, a pyrimidine and pyrimidine?

Mm... Why not??!

It won't. Because, when a purine and purine base pairs that region would be somewhat wide, due to the bulkiness of purines.

If pyrimidine and pyrimidine, that region would be so narrow. Imagine, this will lead to an irregularity in the DNA's structure. It will appear something like this. (Bulky and narrow, bulky and narrow.)

:D :D See how beautiful this irregular DNA is??? :) :) 
This won't be stable.
You know, when DNA is unwound, it will stretch into kms???!!!
Shh.... Don't reveal this secret to anyone. (DNA- Secret of Life) :P :P :)
Happy learning..! Bye. Bye.

Sunday, July 15, 2012

Cassia angustifolia (Senna) as Laxative

Cassia angustifolia is a drought resistant plant. It is a native plant of Saudi Arabia. But, nowadays it is grown widely in India also. It was first grown in India, in Tirunelveli district of Tamil Nadu. Now, it is grown in Tirunelveli, Ramanathapuram ditricts of Tamil Nadu and also in Gujarat and Maharastra. The importance of this plant lie in it’s constituent “Sennosides”. It has Sennosides A, B,C,D. But, only A & B are used as medicines.

English : Indian senna, Tinnevelly senna
Cassia angustifolia
Hindi Sanay, Sana ka patt
Kannada Nelavarike, Sonamukhi
Malayalam Sunnamukki, Connamukki
Sanskrit Svarnapatti
Telugu Nela tangedu
Tamil Nilavarai, Nelavakai

It can be grown even in waste lands, there is no need for frequent irrigation and maintenance. It is one of the major medicinal plants exported from India.
But, it is not used much by Indians and it’s consumption is less in India.

About 180 tonnes of Calcium sennosides is produced in India per annum. Out of 180, 140 tonne is exported.

Generally leaf extract is used for laxative preparation. Leaf has higher sennoside content. The young leaves are found to have high sennoside quantity than the matured ones.

Dosage as laxative: 0.5 to 2 grams.

Researches are going on for increasing the sennoside content of senna by applying stress (Plant tissue culture)

Tuesday, July 3, 2012

Basics of Biology!

Before getting deep into something, we must know the basics. Here we go with some basic questions and their answers.

Question number 1: What are the three major domains of Biological system?

Prokaryotes ; Archaea ; Eukaryotes

Prokaryotes: Example: Bacteria
They lack true nucleus. They have cell wall. They lack membrane bound organelles.
The bacteria which is used mostly are Escherchia coli (E.coli)

Eukaryotes: Example: "WE" - Homo sapiens (Humans)
The eukaryotes are well developed, multicellular. They do not have cell wall (but plants do have, there are certain exceptions). They have well developed nucleus, membrane bound organelles.

Archaea: (False bacteria)
They survive extreme conditions (extremophiles). They were isolated even from volcanic hot springs, imagine, how good they are in surviving extreme conditions. They look similar to bacteria but their metabolic pathways and genes are closely related to eukaryotes.

Question Number 2: Why eukaryotes don't have cell wall? While prokaryotes do?

Prokaryotes are just single cells, there is no need for them to contact or transfer molecules between other cells (but, they do during reproduction, generally not needed). They need not interact with neighboring cells to form tissues.

But, Eukaryotes, they have to interact to form tissues and further organs. That's why, for interaction, Eukaryotes don't have rigid cell wall!

Question number 3: Who discovered Structure of DNA?

I was thinking, it was James Dewey Watson and Francis Crick,  but, two others also contributed, they were Maurice Wilkins and Rosalind Franklin. But, Watson, Crick and Wilkins were only awarded with nobel prize because, Franklin died before that due to cancer.

This discovery of DNA structure was the starting for genetic engineering.

Take Care,
Bye Bye! :)

Monday, July 2, 2012

Professional Ethics!

Personal and professional ethics are different. Professional ethics vary from one profession to other.
Doctors have different, engineers have different ethics. As a technology student, am having a paper "Professional ethics and human values" in my syllabus.

I wondered,
Is it essential to teach ethics? human values? to an engineering student?

What will you answer to this question? Yes? NO?

If yes, you may justify saying, yes engineers and doctors must have some ethics, human values.
I agree, but, is it essential to teach ? Have it as a separate paper? Yes, it's essential, we must have some ethics and this paper gives an idea like, how to approach a social problem, how to deal with the customers.. so on..

But, i won't answer yes! I will answer "NO"
Ya, Ethics and human values are not something which one can obtain by studying a paper and appearing for an exam. As a doctor, he must be having that values in him, he must be so kind at heart, must not be money minded. As an engineer one must be able to deal polite with his clients or must be able to analyse or think about a problem, he must be able to be responsible and must only invent/ discover harmless products.

But, all this can't be achieved only by studying a paper. One must have these ethics. It's must!

Teaching Human values! Oh God, we are in such a bad situation, basic values like love, caring, sharing, non violence are taught as subject to Engineers and doctors(i'm not sure about medical syllabus) at college level! There is no need to teach this. You must have this values or you must had developed it in the childhood itself? isn't it?

Here, they are teaching  human values with books, theories and laws? :( This means indirectly, engineers are loosing their values? In the beginning, when Engineering was budding in India, Engineers were not taught with these values. But, in the middle, they added this in the syllabus. This means, Engineers were found to have least values, and, after that, to make them aware of "The importance of Human Values", this was added to syllabus?

But, even now, this is not going to help. In this money minded world, only the people who are already having some basic human values will follow these ethics, the others won't. The people who are money minded and who never worry about human values, they will read  this paper and write the exam only for getting a B.E or B.Tech and not for getting human values.

Instead, the ethics and human values of an engineer or doctor can be tested with a personal interview by an expert psychologists panel. After a personal interview, B.E., B.Tech., M.B.B.S or any other professional degree can be issued.(Strictly no written exams - because these people will work out previous year question papers and get through)

Haiyo.. Why am I thinking this much? to this extent? :O

This is just my view. Sorry, if it contradicts your views.

But, after all this, I loved my professional ethics class :)

Okay, bye. Wish you good time!