A Butterfly and Biotechnology

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 ...

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 ...

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 g...

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 firs...

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 lik...

Hi, During the last practical exams I shared my experience. (Read here) I shared certain questions with answers which the examiner asked me during the practicals. Even this time, I'm going to share a few questions and answers with you. Here we go, This time I had 2 lab exam (Practicals), number one - Bio informatics and two - Molecular biology. Let us start with the Bio informatics lab. Bio informatics:   In this Bio info practicals, we were provided with two exercises - one must be done using various bio info tools such as Clustal W, T-coffee, BLAST P, BLAST-N, PHYLIP etc., The other exercise is PERL programming! :( :'(  The first exercise using tools is very simple, but, I find the programming, let it be any programming, it's very difficult :'( Even in the model practicals, I couldn't debug the program :( So, I worked really hard to learn the PERL! I was working with my lap for a whole day. And atlast, I got good confidence that I could write my own pro...

Hi, Agarose gel electrophoresis In the previous post I explained how to quantify DNA, and now we can find the molecular weight of that extracted DNA. But, how? Simple, you need to run an agarose gel! Preparation of agarose gel: Agarose gel must be prepared based on the DNA we are going to run. For example: if you are going to run Genomic DNA, you must use 0.6%gel, for plasmid DNA, you must use 0.8% gel, for PCR(Polymerase chain reaction) sample, you must use 1.2% gel. You may ask, why three different % of gels? The answer for your question is: Genomic DNA will be bulkier than pDNA - so, we must use a gel with less percentage, so that the pore size will be greater and the genomic DNA can move easily. In case of PCR sample, the gene amplified won't be bulky, so, it moves fast, very fast in low percent gel, that's why to have an optimum speed we use a higher percentage of gel comparatively. And, when our aim is to separate two sequences with relatively equal size...

Hi, I explained the principle behind the extraction of pDNA in my last post. Extracted the DNA, what next? Let us quantify the DNA extracted. How to quantify? Very simple, all you need is Spectrophotometer. Just take 1 microliter of your DNA extract in an eppendorf, make it upto 500 microliter with nanopure water. (else 1000, or any desired volume) mix it well Now, put this into a quartz cuvette (0.5ml), measure the O.D value at 260 nm. Use blank as nano pure water. Note: The path length of the cuvette must be 1cm. Okay, we got the O.D value now, how to get the concentration of your DNA? It's very simple, do the following calculation. If your O.D. Value is 1, then, the concentration of your DNA sample is 50 microgram/ml so, now, if your concentration = obtained O.D.( let it be 0.5) * 50 microgram/ml * Dilution factor How to calculate the dilution factor?  Simple! here your dilution factor is 500/1 i.e., 500 micro liter contains 1 micro liter of DNA, you can calcu...

Hi, Have a good time :) Last time I had explained you the procedure for "electroporation"  and now with those electroporated cells, you can extract the plasmid DNA. I'm not going to share the exact protocol here, but the mechanism behind the protocol we use in our lab. Generally, the following reagents are used for the extraction of plasmid DNA., 1) STET buffer 2)Lysozyme PUC19 3)RNAase 4)Phenol chloroform 5) Alcohol 6) Sodium acetate If you need the protocol, kindly mail me I'll send you the  protocol. Now, let us discuss the principle behind the usage of each of these reagents. STET buffer - contains Sucrose which helps in maintaining osmotic pressure; Triton X which helps in cleaving the cell wall; EDTA which acts as a chelating agent; Tris HCl is used as buffer. Lysozyme - cleaves the cell membrane and wall; RNAase- As  the name indicates, it cleaves RNA Phenol chloroform (buffer saturated- pH 8) - Phenol denatures proteins, Chloroform pr...

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, you...

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...

Hi, I was thinking deeply, that how to spend my semester holidays. Got an idea of doing a mini project in my department. Let me explain you how to start with a project or how i did start. Here we go... Step 1: Choose or find out your area of interest. I mean the subject your interested or your favourite subject. You may wonder, how do i know, I hate all the subjects! But, don't worry.  Choose the subject you feel more easy than others. Step 2: Read research papers online related to your area of interest. Just google and read random stuffs, you will get an idea. There are many research papers available online ( You can try online magazines for latest research trends) Step 3: While you were reading, something might have interested you. Read much about that topic. Try to think differently, and frame your own project. Note:  The above steps would suit perfect if you are a Biotechnology student. If you find it difficult to start a brand new project, just try to con...

Hi, As I said in my previous post  Viva Voce #1 , my university practical exams are going on, out of the 4 practicals, now I completed 3 :) Today was a day of E.coli, as I got E.coli with all experiments :) Microbio is my favorite favorite subject, and the lab, I love to be in microbio lab with my microbial buddies :) :P Here we go with my microbiology practical exam :) I prepared well for my lab exam yesterday night. I made myself clear with all the concepts behind lab techniques and experiments. Had good confidence :) as this is my favorite lab :) I was given with a question paper saying: Major experiment:  Inoculate 10% of E.coli culture in nutrient broth of ph5 and check the growth rate by measuring turbidity and plot a graph. Minor experiment:  Perform gram staining of the given culture, observe shape, color and interpret the results. :) :) :)))) I got a biggggggggg smile when I saw this easy question paper and started with my experiment :) I clea...

Hello! Here I go with my practical experience :) I'm doing my university practical examination this week. Completed 2 out of 4 practicals :) My mood got spoiled in my lab because of the moody chart!  But, successfully, did the viva voce  good for the 2 exams :) I shared some of my viva voce questions with answers as it might help someone searching for some answers (as I do every time, the day before exam) Chemical Engineering laboratory: I was given with a question : " Draw Moody chart for fluid flow through non circular pipe" with some specifications of pipe diameter. I got shocked, as I don't know what's a Moody chart! :( :( Non circular pipe?!!! Still again a great confusion! But, I managed to guess what was that non circular pipe! It is nothing but, "Annulus". We use to say " Fluid flow through annulus" , so, I got little confused with the twisted question! Then, the moody chart.. I didn't bother about that. I did the expe...