BIL 250 Lecture Notes - Lecture 8: Cytometry, Cosmid, Fosmid
3 Oct 2016
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Isolate your dna from the organism- dna extraction: cut dna to a certain size with restriction enzymes, splice each piece of dna into a cloning vector to create a recombinant dna molecule, transform recombinant dna into the host. Use detergent to break up the cell. Use proteinase k to digest proteins bound to dna. Use a round disk of silica, porous so dna can pass through it unless the dna is precipitated with ethanol. Water will help it pass through when you are ready to collect it. This helps make clean cellular dna that does not have debris. If you add too much material, your yield decreases. Cold temperatures, not taking it out of the freezer too much larger dna fragments. Manipulating the dna, warmer temperature smaller dna fragments. Adds methyl groups to prevent it from cutting its own dna. Cutting with different enzymes gives you blunt ends or different overhangs.
