1. The same restriction enzymes are used to cut desired gene and plasmid at a specific location.
2. The ends are "sticky", as they are single strands that combine together and glued by ligase, forming phosphodiester bonds.
3. Complete digestion means there are enough REs to cut at every restriction sites; Incomplete digestion means not enough to fully cut at all the sites
4. pBluescript is an engineered plasmid with multiple pre-arranged enzyme cut sites.
PCR (Polymerase Chain Reaction)
5. Materials: taq polymerase (heat resistant), single-stranded DNA primer, nucleotides and ligase
6. Heating denatures DNA; cooling allows primers to base pair to their complementary strands; taq polymerase attach at each priming site and synthesize a new DNA strand.
7. The targeted fragment is produced ONLY after third cycle, then is exponentially grown.
Gel Electrophoresis
8. Shorter DNA fragments move down the gel to the positively charged anode (since DNA is negatively charged), and larger DNA fragments remain at the top.
RFLP (Restriction Fragment Length Polymorphism)
9. DNA fragments are cut by COMPLETE digestion by multiple REs, then undergo gel electrophoresis.
DNA Sequencing
10. DIdeoxyribo-nucleotides incompletely digest the DNA fragments, terminating the sequence at each individual nucleotide (ddATP will incompletely digest at every adenine sequence).
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