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 denature and separate proteins

For building a phylogenetic tree, you need more than 2 sequences for both multiple sequence alignment and building a tree using ClustalW.  Phylogenetic Tree in bioinformatics represents the evolutionary relationship between species.  Follow these steps to build a tree: These steps are for building a phylogenetic tree for p53 of 4 different species (Homo sapiens, Mus musculus, Rattus norvegiccus, Danio rario). (You can consider the protein or dna of your interest to find the relationship between any number of species).  1) Go to NCBI homepage and select Nucleotide. And type in the search box "p53" and you will get number of hits for this query. And a number of entries for p53 in different species will be displayed. 2) Consider 4 different species (example: homo sapiens, mus musculus, rattus norvegiccus, danio rario). and retrieve FASTA format sequneces for those 4 species and paste them in a word file one by one.  3) Open https://www.ebi.ac.uk/Tools