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.

[Read More....]

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. 

[Read More....]

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. 

[Read More....]