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Obtaining of tumor tissue sample (Namely liver) from organism, If cell…

- - - - Once cell have reach 50-70% confluency, the cells can be taken for preparation for cell passaging, where we split the quantity of cells into 2 or more different culturing flask for further experiment
        
        Once cell have been passaged, the cells are cultured again to further check
        
        Once sufficient cells have been cultured, they are injected into mice to observe for growth of cancer
        
        If cancer growth is observed, the mice are dissected to remove the
        
        Cells are split with some being on the culture flask and some being placed on the 96 well-plate for other experiments such as drug treatment with Sodium Selenite, Selenocysteine and Erastin
        
        After leaving the cells in the well plate and placed in the incubator, the plate is taken out for treatment of cancer cells with these drugs. The plates are then left in the incubator for 72hrs overnight
        
        After 72hrs has past, the plates are then taken out to fix the cells in place and prepare for SRB assay which helps to quantify the amount of cells that are present
        
        The cells are then taken for cell counting using Countess (TM) II FL automated cell counter.
        
        To do this, trypan blue of 10ul is added into tubes and this are fluorescence dye which will be able to enter those dead cells and give off blue signal that the cell counter machine can pick up with specific wavelength.
        
        Based on what the machine displays, green colour represents live cells while red colour represents dead cells
        
        The recommended amount of cells should be at least a million (n x 10^6)
        
        Each well plate should have 5000 cells/100ul. Calculation of amount of cells required is as followed.
        50 cells/ul = 50000 cells/1ml (50000 x 1000/1 x 1000)
        n x 100 (remove d.p.) / 5 [(n x 10^6) / 50000]
        
        The remaining amount that have not been added into the wells are then taken for freezing
        
        After the cells are frozen, the cells are then taken for DNA extraction. MiniPrep Kit is designed to contain all of the necessary reagents required for extracting the cells.
        There are resuspending buffer, cell lysis buffer and neutralization buffer.
        Resuspending buffer (FADP 1) contains RNase-A that will help degrade RNA that can contaminate with the results since RNA as smaller and not as heavy/big as DNA.
        Cell lysis buffer (FADP 2) contains detergent and other chemicals that will help lyse the cells.
        Neutralizing buffer (FADP 3) is then used to neutralize the lysate by increasing/decreasing pH to neutral level
        The tubes are then centrifuged to pellet the cellular debris to the bottom of the tube. The supernatant are then extracted and transferred into a column to extract DNA
        
        Washing buffer is added to precipitate the DNA and the rest are then centrifuged with a column to collect flow-through.
        Centrifugation at this point is to further purify the sample such that there is no contaminant aside from DNA only