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- restriction enzymes to cut
- Ligase to stick together
- Phosphatase --> phosphate groups
- 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.
- Cas9 = targetable restriction enzyme. really easy to make new pieces of DNA. Can have 20-nucleotide recognition sites
- Site-directed mutagenesis: you can get rid of recognition sites using silent mutations. Melt to separate into two circular single strands, then add primers which aren't direct matches to introduce the silent differences in nucleotides.
- IDT IDT
- -primers <60 BP. Bead-stick on nucleotides one at a time then drop it off the bead to produce your primer. single stranded and short
- gBlocks: double stranded and long up to 2.5kb overlapping DNA strands in a block.
Ligase links the groups together-
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