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MOL BIO - Coggle Diagram

- - - - Tetracycline resistance
        
        Ampicillin resistance
      - Plasmid is cleaved at the Ampicillin resistance gene (by PstI)
        
        Foreign DNA is then ligated to the plasmid where the Ampicillin gene was, effectivly making it not work
        
        This plasmid is then transformed into bacterial cells and allowed to replicate
        
        Due to the nature of plasmids not all the cells replicated will have the pBR223 plasmid and so we need to find which ones do. To do this we first transfer the cell to an Agar Plate w/ tetracyclin
        
        By transfering these cells to tetracycline we are already removing the cells that don't have resistance to tetracycline via the plasmid we're looking for. So by transferring them we are removing just the regular joes from the cell that could potential have our plamid (due to them not dying as they have resistance
        
        These cells are then transferred to two different agar plates to determine whether they have the specific plasmid we want + our foreign DNA
        
        Agar plate + tetracycline (same as before really)
        
        2 more items...
        
        Agar plate + tertracycline AND ampacillin
        
        2 more items...
        
        The cells from the bubble above are taken from one colony onto a small needle and split amongst both plates below. These dots are from the same original colony so they all should be the same cell type (w/ or w/out the plasmid) and to make sure we know they're from the same colony we dot them in the same place on both plates
  - - - Colonies of the cells you want to test are transferred form their Agar plates to a Nitrocellulose paper
        
        These cells are then treated with alkali to "kill" the cells and effectively remove all of the cell material leaving just the DNA on the nirtocellulose
        
        DNA sample of the DNA we want is radiolabled and added to the colonies. The radiolabled DNA binds to the DNA from cells, IF it is similar if not the same.
        
        These are not visable to the naked eye, however can be seen under X-Ray
    - - Chromosomal DNA is cut up via restriction enzymes (link)
        
        This DNA is then tested along with sample DNA inside an agarose gel, in which gel electrophoresis is carried out.
        
        The fragments are then seperated via gel electrophoresis and both DNAs are radiolabled and can be inspected under and X-ray
        
        Thus, if the DNA sample match the chromosomal DNA then you have a match and can successfully ID the DNA
    - - DNA Samples are separated (via head) and the two strands are then separated into 4 test tubes and a specific primer is annealed to the two strands in all 4 test tubes. Along with this to each tube a bunch of nucleotides are added along with a base specifc ddNTP, (ie ddNTP for A, ddNTP for T, etc), one type of which is added to each tube, ie( a tube for ddNTP for A and another tube for ddNTP for T). Here Replication enzymes are added too
        
        The strand are replicated and randomly stop when the DNA poly picks up a ddNTP instead of a regular base (dNTP). Cause the ddNTP doesn have the OH in the 3' terminal. Due to this the fragments all differ in length but each tube has the same ddNTP base pair at the end of their strands
        
        Gel Electrophoresis is carried out using Polyacrylamide Gel to sequence the strands (all four test tubes). The strands move up and are seprated by their size
        
        This seqence is then read upwards from the bottom of the plate. This will give you the complimentary sequence to the sample you were testing
        
        Polyacrylamide Gel is used in place of Agarose gel due to it's higher resolving power and can actually seperate DNA strands by one bp