February 9, 2011

Ampicillin


Ampicillin is one of the most widely used antibiotic in molecular cloning, for the selection of transformants. It belongs to the penicillin group of antibiotics i.e. beta-lactam antibiotics. The only difference between penicillin and ampicillin is the presence of amino group in the latter.


            Unlike penicillin which is effective against only gram positive bacteria, ampicillin is also effective against gram negative bacteria, e.g. E. coli. The amino group present in the side chain of the ampicillin renders it to penetrate the outer membrane of gram negative bacteria and hence enter into it. Therefore, it was one of the first broad spectrum penicillins to be used.

February 5, 2011

7 Factors affecting expression of heterologous proteins in E. coli

The gram negative bacterium, E. coli is a versatile and valuable organism for the high-level expression of heterologous proteins. This is mainly because the bacterium is easy to genetically manipulate, inexpensive to culture and expression of recombinant proteins is comparatively fast.



             However, in spite of all these features, all recombinant proteins are not efficiently expressed in this organism. This may be due to certain factors including the affinity tag used, size and source of the heterologous protein, codon biasing, E. coli strain used, culture conditions and protein degradation affecting the expression of recombinant protein. Here you will find the details of these factors affecting the expression of foreign proteins in the bacterium. 

February 1, 2011

RNA isolation Protocol


Isolation of the high quality RNA is the first step for numerous molecular biology experiments. These experiments can be RT-PCR, Real-Time PCR, cDNA library construction, Northern blotting, RNA mapping, in vitro translation, etc. Here, in this article, we will deal with detailed protocol of RNA isolation.

            The biggest rival of RNA during the extraction procedure is RNases. In RNA, hydroxyl groups are present at 2' and 3' positions of ribose sugars. This makes RNA much more chemically reactive than DNA. Therefore, contaminating RNases easily cleaves the RNA. 

January 30, 2011

9 Factors affecting DNA extraction from agarose gel


           Agarose gel electrophoresis is a method of choice for the identification, purification, and separation of the DNA fragments. DNA fragments from the gel are routinely extracted for various downstream processing. These include, cloning, radio-labeling, in vitro transcription, microinjection and sequencing of the DNA molecules.



            
           Gel extraction of DNA fragments is mainly done to remove proteins and salts that incorporate from certain reactions. Therefore, in order to use the DNA fragments for downstream processing, these components musts be removed. For example, a PCR amplification or restriction enzyme digestion reaction contains factors which inhibit further applications of the DNA fragment.

January 27, 2011

Choosing the right DNA polymerase for your PCR


Success of the polymerase chain reaction (PCR) largely depends on the choice of the appropriate DNA polymerase. DNA polymerase is one of the major components needed for setting up a PCR. For PCR, a thermo-stable DNA polymerase is essential, so that it can endure higher temperatures during the cycling conditions. Therefore, thermo-stable DNA polymerases serve as a key player in the current methods of DNA amplification and sequencing.


Based on their amino acid sequences, DNA polymerases are categorized into six families: A, B, C, D, X and Y. Thermophilic enzymes are found in all the six families. Same reaction is catalyzed by all DNA polymerases, that is, adding nucleotides to the 3'-end of the DNA primer to synthesize the new DNA strand complementary to the template DNA. The thermo-stable DNA polymerases synthesize DNA in a template-directed manner, and require primer-template hybrid to begin the synthesis.

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January 24, 2011

Three commonly used affinity tags for protein purification

In order to purify heterologous proteins from various hosts, affinity tags are extremely capable tools. In the recent years affinity tags have become highly popular tools for protein purification mainly due to following reasons. They provide high level of purification of recombinant proteins from crude extracts, mostly in a single step. Second, they provide mild elution conditions, thereby, do not interfere with the structure and hence the function of the purified proteins. In addition, affinity tags allow a variety of proteins to be purified using easy procedures.



            Affinity tags are available as expression vector systems having multiple cloning sites (MCS) for cloning the gene of interest towards the N or C-terminal of the tag. Now-a-days, a variety of affinity tags are available for the purification of recombinant proteins. Each tag has its certain advantages and disadvantages. Here, you will see the properties of the three commonly used affinity tags: His-Tag, GST-Tag and MBP-Tag.

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January 22, 2011

RNA Isolation: Principle and Procedure


In order to perform various fundamental molecular biology experiments, the first and most crucial step is isolating high quality, intact RNA. These experiments include Northern hybridization, RT-PCR, RNA mapping, nuclease protection assays, in vitro translation, cDNA library construction and so on.


 In a cell, 80-85% of the total RNA is contributed by ribosomal RNA (rRNA). However, messenger RNA (mRNA) is only 1-5% of the total cellular RNA. 

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