BIOMAG

In bacteria, the Plasmid is present in the cytoplasm. To get the plasmid DNA out of the bacteria, its cell membranes must be disrupted in order to get the Plasmid DNA in the extraction buffer. There are three main solutions involves in the procedure.  Solution I : It contains Glucose, Tris and EDTA.. Glucose provides the Osmotic shock which leads to the disruption of the cell membrane. Tris  is buffering agent and maintains constant pH 8. EDTA is used to protect the Plasmid from endogenous nucleases by chelating the magnesium ions.  Solution II : it contains NaOH and SDS. This is an Alkaline solution. It is used to disrupt the cell and NaOH also denatures the DNA into single strands.  Solution III : it contains the acetic acid and potassium acetate. Acetic acid is used to neutralize the pH. and Potassium acetate is used to precipitate the chromosomal DNA, proteins and other cellular debris.  Phenol/Chloroform: It is used to denatu...

Extraction of chromosomal DNA from eukaryotic cells is the common procedure in molecular biology experiments and forensic analysis. Chromosomes of eukaryotic cells are located within the nucleus of the cell. Each eukaryotic chromosome consists of a single DNA molecule wrapped around the histone proteins. In order to get the chromosomal DNA out of the cell following steps are used.  1) Cells are treated with detergents such SDS (Sodium Dodecyl Sulfate), Triton-X-100 or Tween in order to break open the cell. These detergents will break the plasmamembranes and nuclear membrane of the cell and cytoplasmic material & proteins are digested. (The membranes are dissolved using detergents that solubilize the phospholipids that make up these membranes).  2) Once the membranes are dissolved, with the help of centrifuge, the cell lysate is formed. This cell lysate contains the cytoplasmic material, organelles and nuclear material and chromosomes. 3) The proteins...

1.       Spin down 50-100 ml well-grown bacteria, 3600 rpm,15min. 2.     Resuspend bacteria with 20 ml Buffer S, immediately add 100 µl ProteinaseK (10 mg/ml). Vortex to make sure no chunks. 3.       Add 2 ml of 20% SDS, mix gently by inverting. 4.       Incubate the mixture at 65 o C for 1 hr with inverting every 15 min. 5.     Add 10 ml of phenol and 10 ml of chloroform, mix thoroughly by inverting for 5 min, spin at 3600 rpm for 20 min. 6.       Transfer supernatant to a new tube, add 0.6 volume of isopropanol. Mix gently by inverting. You will see cotton-like genomic DNA. 7.       Hook out the cotton-like DNA to a 1.5 ml tube; wash with cold 70% ethanol. 8.      Dry DNA at RT, dissolve DNA in 500 µl H 2 O (50 o C or 4 o C overnight). 9.      Add 5 µl DNase-fre...

Gene Cloning Technique in Molecular Biology Field Involves the following steps: 1.a) Isolation of Genetic Material and Gene Sequence of Interest.  First, cells are lysed using detergent or lysozyme enzymes which disrupts the plasma membrane and release the genetic material along with the macro molecules such proteins and RNA molecules. Cell contents are then treated with protease to disrupt the proteins and RNase to destroy the RNA. Cell debris are then pelleted using centrifuge and supernatant containing DNA is transferred to a fresh and clean tube. A proper amount of Ethanol is added to this supernatant and precipitated using centrifuge. Supernatant is discarded and the pellet which has DNA is suspended using a proper suitable buffer.  Primers are designed for the specific gene sequence of interest which will be used for the cloning procedure.  Gene sequence is then amplified using PCR (Polymerase Chain Reaction) which will yield in many copies of...

The Recombinant DNA (rDNA) Technology refers to the process of joining foreign DNA segments or DNA segments of interest from different sources and introduced them into a host organism for example a bacterial host. This technology enables the construction of new combination of genes or genetic material to be artificially constructed in the laboratory conditions. These rDNA molecules are introduced into the host cells where they can multiply which is to create, replicate and investigate the recombinant DNA molecules.  The best and first example of recombinant DNA technology is Human Insulin produced by bacteria, which was first approved by the FDA in 1982.  A typical Recombinant DNA experiment involves following main steps. 1) Isolation of DNA segments of interest usually gene sequences. (Sequences which are to be manipulated or used and needs to be cloned).  These selected DNA segments are called as Inserts.  2) A specific Vector is selected whic...