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Showing posts from July, 2012

Electroporation (Transformation)

Hi, 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. 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 electroc

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&

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

Hi, 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

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'

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