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...

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.e...

This is a practical experiment steps to carry out BLAST search of a given nucleotide sequence.   (You can either watch the video or go through the steps mentioned below the video).  Follow These Steps: Open NCBI home page http://www.ncbi.nlm.nih.gov/ Click on the text box which shows all databases and change it to "Nucleotide. In the search box, type name of a protein or gene whose sequence you would like to retrieve. For example: Human Insulin. Click on Home sapience at the right corner and you will get the list of all related Homo sapience.  Select whichever you want and click on it.  You will get the data in the format of Genbank. Since BLAST accepts only FASTA format, you need to change GenBank format to FASTA format by clicking FASTA. (just below the heading line).  The new page will appear where you will get the FASTA format of the nucleotide sequence.  Copy that sequence along with code present in the format. ...

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...

Protocol:  Preparation of Samples for Cell Cycle Analysis by Flow Cytometry: 1) Suspend approximately million cells (10^6) in 1mL of phosphate-buffered saline solution (1x). And with the help of pipette, gently aspirate about 7-8 times, to make the single cell suspension.  2) Take a centrifuge tube and add 4mL of 70% ethanol. (70% ethanol should be one ice before using). and add 1mL of cells in PBS to this tube. Keep the tubes on ice for minimum 2 hours at 4℃.. This step will fix the cells.  ( cells can kept at 4 ℃ for a week).  3) Centrifuge the ethanol fixed cells for 5 minutes at 300g. Carefully take out the supernatant. Do not disturb the pellet.  4) Add 5mL of cold 1x PBS to this pellet and suspend the cells using pipette.  5) Centrifuge the tubes at 300g for 5 minutes. and discard the supernatant. Again do not disturb the pellet.  6) if required, step 5 can be repeated for one more time.  7) Add 1mL of Propidium Iodide (PI...

Reagents:  Release Buffer :Phosphate-buffered Saline,  pH 7.4                           10mM EDTA FACS Buffer :   Phosphate-buffered saline,  pH 7.4                        5% FBS (Fetal Bovine Serum)                        0.02% sodium azide                        Ice Cold. If you are going to fix cells:                        Phosphate buffered saline                        2% formaldehyde (w/v)  (from stock 37%) Protocol : (This procedure is for HeLa cell line which is adherent cell line, they need to be detached from the cell culture plate to proceed with flow cytometry experiment p...