BIL 250 Study Guide - Midterm Guide: Low Copy Number, Cloning Vector, Puc19
15 Nov 2016
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Sds (detergent)- break up cell membrane & organelles. Salt- lyses cell and binds dna strands together. Proteinase k- digests proteins bound to dna (remove euk chromatin) Recognize specific bp sequences (restriction sites) & cleave (hydrolyze phosphodiester bond) Cut happe(cid:374)s (cid:271)et(cid:449)ee(cid:374) 3" (cid:272)ar(cid:271)o(cid:374) of 1st nc and phosphate of next. Restri(cid:272)tio(cid:374) frag(cid:373)e(cid:374)t e(cid:374)ds: 5" phosphates & 3" h(cid:455)dro(cid:454)(cid:455)ls. Splice (ligate) into cloning vector = recombinant dna molecule. Transform (cloning vector & dna fragment) into host. *cloning vector = artificial dna molecule capable of replicating into host. Protect bacteria against bacteriophages by cutting up viral dna. Via methylation, bacteria modify own restriction site to protect dna. Names: usually begin with 3 italicized letters (ecori- e. coli ry13) Frequency = (1/4)n n= number of nucleotide pairs in restriction site. Sticky ends useful: compl sequences can be joined directly by ligase. Bacterial plasmids: naturally occurri(cid:374)g, s(cid:373)all (cid:862)satellite(cid:863), circular d extrachromosomal dna.
