BIOL 5060 Lecture Notes - Lecture 7: Transferase, The Technique, Molecularity
Document Summary
Gateway plasmids series- combine entry plasmid (like traditional cloning) to put sequence of interest into it all kinds of specialized plasmids by recombination: highly efficient. We cant do this until we know the sequence of the gene of interest. Steps 1-3: most common inserts= genomic inserts or something that represents the transcribed region through cdna (complementary) Genomic dna: how are these ^ produced, genomic dna is made by 1. Partial digest with a restriction enzyme to generate somewhat random fragments (if we use a limited amount of. Sau3ai or give limited time of digestion, we get limited fragments) 3. Gel electrophoresis (bands allow us to select approximately how big we want our things to be) size selected molecules by cutting out slice and eluting dna (limits to how big inserts can be) 4. Sau3ai partial digest 2. molecules with gatc 5" overhangs 4. Treat with klenow + dgtp and datp : add ag nucleotides (2 nucleotides prevent circularization) 6.
