MBIO 4600 Lecture Notes - Lecture 26: T7 Rna Polymerase, Restriction Enzyme, Flanking Maneuver
25 Dec 2015
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Second recombination: outcome depends on crossover event, screen by pcr, southern blot, or sequencing, mutant or wild-type. Degrades t7 rna polymerase in absence of iptg. Conventional method (cid:1: pcr left flanking region from genomic dna with engineered restriction endonuclease sites. 6-8 bp overhang necessary: digest/ligate into cloning vector (circularize), transform, and screen colonies, pcr right flanking region from genomic dna with engineered restriction endonuclease sites. Right flank of gene you want to get rid of: digest/ligate into plasmid construct with first insert, transform, and screen colonies. Plasmid with 2 inserts (left and right flank: pcr with engineered restriction endonuclease sites (or digest) antibiotic resistance marker cassette. Plasmid with antibiotic resistance marker cassette: digest/ligate into plasmid construct, transform, and screen colonies. Plasmid with antibiotic resistance marker cassette, and: cut out construct and re-ligate into suicide plasmid, transform, introduce plasmid into recipient strain to generate gene right and left flanks into host cells, and screen replacement.
