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 ...

Though there are several protein assays available, the most preferred one in many laboratories is "Lowry assay". It is effective in the concentration range of 0.01 mg/ml to 1 mg/ml. And, as an additional info, the paper published describing the procedure and principle of Lowry Assay is the most cited paper in the scientific history. (Feeling like, "Wow! I want to publish one to compete with Oliver.H. Lowry"???  :P) Why Lowry?   Though there are several other protein assays, mostly Lowry assay is used in many laboratories. The reasons for preferring Lowry are: sensitivity of the assay, highly reproducible, cost effective, easy to perform. Biuret assay is generally used for higher protein concentrations like tissue samples but, Lowry for less concentrated samples and hence used in most of the molecular biology laboratories where there will be need for assaying comparatively less concentrated protein samples (in most cases where we attempt to produce enzymes). Oth...

It's been a long time, since I wrote a post here. Finally, final year of my course and so very busy :) with books! And now, got time to share with you. As you know already (don't know?, then get to know :P ), I use to have favorite subjects in each semester, third semester it was microbiology, then in fourth semester, it was cell biology, fifth was molecular biology, sixth was genetic engineering and now i'm in seventh semester, but, you know what, I don't have a favorite subject!!! Don't worry that I lost interest, instead, I got 3 favorite subjects - Immunology, Animal biotechnology and Plant Biotechnology! It's awesome this time to have more than one favorite subject and here I'm gonna tell you something about, cre-lox system which is most widely used for making modifications (mostly deletions). These modifications can be done at a specific tissue alone by using a tissue specific promoter i.e., you can selectively knock out a particular gene in partic...

Let it be any disease, ranging from a fever (which is the symptom of many diseases) to HIV, diagnosis at an early stage is very important for giving specific treatment.Whenever I go to a doctor, he asks me things like, stomach pain, fever, body pain, head ache, running nose, throat pain etc., then, he checks pulse, then he uses his stethoscope, he then gives all the possible antibiotics! Yes, really, this happens in most cases. Then, if my problem is not fixing up in a week, he'll ask me to take a blood test, this, that, and, all! Nothing wrong in this, because he can't ask everyone coming to him to take blood test at the very first sitting, then, no one will visit him back saying he sucks out blood all the time! But, it is okay to have a blood test after a week, but, not after a month! Diagnosing a disease after it had reached a severe stage is comparatively less significant than diagnosing at an early stage. So, how to diagnose? Antigen - Antibody! Antigen and Antibody ...

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 ...

Oral cancer refers to cancer in any part of your mouth like tongue, lips, palate etc., Cancer may be due to various reasons like irritation due to continuous alcohol consumption, smoking, poor oral hygiene etc., And it can also happen due to Human Papilloma Virus (HPV). Human Papilloma Virus is a type of non enveloped virus (Papilloma) that can infect humans. They are classified generally as "High Risk" and "Low Risk" types. The high risk type HPV 16 is found to cause oro-pharyngeal cancer. Diagnosis: Generally biopsy is done where a small part of your tumor is removed and observed under microscope. The biopsy can be just cutting a small portion or it can also be done using brush for collecting the cells. After confirming the abnormality using this microscopic observation, further analysis is done for finding out the type of HPV which caused the oral cancer or to find whether the cancer is just because of alcohol/smoking. This further analysis can be d...

It was 24th December I believe! I just saw the details of the Indian Academy of Fellowship only a week before the last date. I decided to apply for it and just applied in a hurry actually, yes, it was really a hurry! I was done with everything and sent the application. I was waiting for the release of selected list of candidates from the first of March. They updated the selection list everyday but my name was not on the list till 25th of March! I used to go to the IAS website everyday and check the list, but, my name was not there in the selected list of candidates. I lost all my hope and decided that I won't get selected. But, it happened! I received a mail from the Academy after 25th of March! I was happy to the core, jumping up and down. Because, I was never out of my town from my child hood all alone. And, this fellowship was something really precious for me! I believed that this would change my destiny to a better place! I was happy till my father said "no" to th...

We are going to discuss here about three different typing! They are genotyping, phenotyping and karyotyping. Before learning about them let us learn what is genotype, phenotype and karyotype! Genotype, phenotype and karyotype Genotype refers to the genetic make up of an organism. Generally the genes of an organism. The genotype of an organism can be represented as BB or Bb or bb based on the gene. If a person is having two recessive CFTR genes, then he will be getting cytic fibrosis. Genotype of an organism has also effect over the phenotype. Thus, genotype is representing the alleles of a gene in general. Genotyping is generally done based on PCR or hybridization. Phenotype refers to the visible characters like structure, color and also the biochemical characters. This is based on the phenotype. Phenotyping can be done using biochemical assays. Karyotype refers to the number of structure of chromosomes in an organism. karyotyping is done by staining and visualising the cells...

As a biotechnology student whether I'm taking care of my meals plate or not, I have to take good care of my petriplates. I wash my petriplates better than my meals plate! Yes, we never autoclave and sterilise our meals plate, but we do for our petriplates! We ( I mean me and my friends) enjoy cleaning and autoclaving our petriplates in our  department media room. And you know, my friends always complaint that I'm never accompanying them in cleaning. Yes, I'll go, wrap my petriplates, put it in autoclave, but I find it very hard to wash! Always, I do autoclave, but, never wash after the autoclaving, I'll give that easy job to my friends :P I love my petriplates! Yes, I love them a lot. But, how do I say that I love my petriplates? I love them because I'm always with them. I love them because, even whether I do complex Genetic engineering or simple Microbiology, I always need them! And the final reason for loving my petriplates is they are so cute, especially,...

I learnt genetic engineering, and of course, about plasmids. But, when I was questioned about the types of plasmid, God, I was blinking! :( I had known plasmid only as a vector most times, I was aware of the types of vectors but, types of plasmids? :( Okay, It's not a great shame, I learnt it now! Let me share here about the types of plasmids, just what I learnt. Based on the functions of a plasmid they are classified as, Resistance Plasmids: These are the plasmids which have resistance genes. For example the gene coding beta lactamase which offers resistance against beta lactum antibodies like ampicillin. This is an important thing which is used while performing cloning experiments for checking the transformation. This was called as "R Factor" before the discovery of plasmids. Fertility plasmids (F Plasmids): And, these type of plasmids they have important genes called tra genes, never wonder, tra genes are transfer genes which are essential for the non sexua...

Fail??? "Oh, come on, you should not say like this", is this what you are feeling, after looking at this title? If so, don't worry, I'm not meaning this in a negative way as you think. "Everything in life has ups and downs. What about this field of Biotechnology? It's not preferred by many students. Yes, I agree, it is not like the IT field, you can't find a perfect job after completing just your under-graduation, even if you find a job, that won't pay you much. So, obviously, people will go for areas which would give them a bagful of money. "You think, Biotechnology is worthless then? you say, you won't get paid?", is this your next question? If so, I'm not saying that. Biotechnology, here, you can achieve heights only after years of enthusiasm and work in your laboratory. It's not the matter of earning money. This field is for people who want to achieve or discover something which is in your own body, in plants around yo...

Southern blotting is used for finding specific DNA sequence from a DNA sample by using the combination of techniques like  gel electrophoresis, capillary electrophoresis and hybridization. The DNA from the gel (after gel electrophoresis)is transferred to a nitrocellulose membrane and hybridization analysis is done for finding out the sequence of our interest using a probe) Let us discuss here the steps involved in doing Southern blotting. The steps are 1) Preparation of Sample 2) Restriction of the Sample 3) Gel Electrophoresis 4) Pre-treatment of the gel 5) Blotting 6) Hybridization 1) Preparation of Sample: (plant/animal/bacterial) For tissue or blood samples the DNA could be obtained by treating with SDS (Sodium dodecyl Sulphate) If we are going to have the DNA from a bacterial sample, there are separate protocols for extracting genomic DNA and pDNA . For extracting DNA from plants, CTAB (cetyltrimethyl ammonium bromide) is used. This CTAB binds with the DNA and h...

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...

E.coli VS Plants! As an undergraduate of Biotechnology, I know about cloning . I know about cloning a gene which codes for a human protein into a bacterial cell, E.coli, most times. I also know about producing transgenic plant varieties. But, I don't know about cloning a human gene into plant! I came to know about cloning a human gene into plant and producing the protein in plants from the workshop session of AVIDADHAM'13. Let me explain something about this! As you may know, eukaryotic gene has both introns and exons. So cloning a eukaryotic gene into a prokaryote will have lots of problem like splicing and post translational modification. Generally for cloning eukaryotic gene into a prokaryote like E.coli, we do follow bottom up approach where we use mRNA to synthesise cDNA, to remove the noncoding introns. But, even if we remove the introns there will be problems with post translational modification. So, to avoid this, we are cloning our gene from human into pla...

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...

We all know that we can cut and engineer our DNA or DNA of any other organism. But, so far the methods used for DNA cutting are not precise. Available techniques: By the older method, the genes are added into the cell and they get inserted into the genome of the cell in a random manner. This can’t be used when our aim is to engineer the DNA at a specific site and here we can’t also replace the already existing gene with our gene. There is homologous recombination method, but unfortunately natural recombination won’t occur in cells that easily! There are also methods which use Zinc fingers for delivering nucleases which cuts the DNA, but, this is really hard, because nuclease can’t target every possible DNA sequence. This is costly too. TALENs – Transcription Activator Like Effector Nucleases – cut the DNA at specific location. But, what to do, this is also costly! Our new method Here, we are going to use the already  existing proteins of bacteria...

We all love sweets, isn’t it? From a cute baby who had just started feeling and enjoying the tastes to 100 year old people, love sweets! Oh, I could hear you, yes, Diabetic patients can’t enjoy sweets. But, they too love to have sweets. Even, my father is diabetic and he uses to eat sweets hiding from my mom! Okay, what for that sweet love, you are asking? Nothing wrong in that, but, if you are crazy about the bottled drinks, then, I’m sorry, you must start worrying! L Aspartame – is an artificial sweetener which is used in most of the bottled drinks. And, that is an approved artificial sweetener by FDA (US Food and Drug Administration – which tests and approves all food and drug items before they are marketed) “It is approved by FDA, then, why we must worry?”, is this your next question? Let me answer you! Aspartame is formed using two aminoacids – L-Phenyl alanine and L aspartate. This when broken down during metabolism in our body produces – phenylalanine and hence ...