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DNA TECHNOLOGY, isolation of plasmid DNA - Coggle Diagram

- - - - isolation of total cell DNA from gene donor (bacteria, plants or eukaryotes) MAKE A FLOWCHART
    - - Restriction enzyme cleaves both the plasmid and digests the donor genome
        
        PCR primer: incorporate restriction site/bacterial promoter?
        
        Or choose restriction site that allows the gene of interest to be inserted aft a bacterial promoter?
    - - via DNA manipulative enzymes
        
        nucleases (cuts DNA/RNA)
        
        exonucleases (3' -> 5' is proofreads, 5' -> 3' replaces downstream nucleotides)
        
        endonucleases
        
        restriction endonuclease
        
        ligase
        
        facilitates formation of phosphodiester bonds (both ss or ds DNA/RNA)
        
        polymerases
        
        DNA polymerase, reverse transcriptase
        
        most require primers
        
        Klenow fragment
        
        modifying enzymes
        
        alkaline phosphatase: prevent self-ligation
        
        polynucleotide kinase: transfer beta phosphate from dNTPs to 5' carbon of the DNA fragment
        
        terminal deoxynucleotidyl transferase: template-independent polymerase, can convert blunt ends to sticky ends via homopolymer tailing (can't restrict length, so must use Klenow fragment to fill in gaps)
    - - electroporation
      - heat shock
    - - selection
      - screening (blue-white xgal/GFP)
      - fingerprinting (southern blot, restriction enzyme digest)
  - - - components of bacterial cells + functions
        DRAW A DIAGRAM AND LABEL
      - Gram positive and gram negative nature affects
        
        what's the procedure + purpose of each step?