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  1. restriction enzymes to cut
  2. Ligase to stick together
  3. Phosphatase --> phosphate groups
  4. PCR: heat double stranded DNA at 94 degrees for 30 secs and the strands come apart. Unique flanking primers. We need the primers to have the same melting temperature, say 60 degrees (geneious can estimate this), then cool mixture to 58 degrees. Then use Taq polymerase to bind to the primers and march down the single stranded DNA adding the complementary nucleotides. (72 degrees- primers don't melt because even at 58 degrees Taq starts adding more nucleotides and thus the dissociation temperature increases.) Choosing extension time is important - Taq has a finite speed. Repeat until you have enough. Exponential increase until you run out of primers and nucleotides. Polymerases get denatured over time. 

Ligase links the groups together- 

Issues with old way: the complementary ends on the plasmid could stick to eachother. So use 2 different restriction enzymes so the restriction sites are different for each end so it can't bind itself. 

. Use selection. Treat with enzyme : each end of plasmid contains part of gene toxic to E Coli, so if they stick together E Coli dies. Select using multiple resistances. 

Screen using GFP. Add LacZalpha marker. It is a complex that turns galactose into lactose and glucose (or the other way round?) Put on plate with X-gal which turns blue when LacZ is floating around. The colonies which aren't blue are the ones we want- the LacZ should've been split apart due to the gene being inserted.