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10 Cards in this Set

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sanger method
uses DNA poly to incorp new monomeric units on 3' end
dNTP's(nucleotides) added and the two phosphates are removed to give energy for rxn and 3rd phosphate is incorporated in the backbone of new strand...syn in 5'-->3'
spin column
allow plasmids to be purified by adsorption. The plasmid DNA is adsorbed to DNA and leaves RNA and prot

then plasmid is ppted w/EtOH and dissolved in Tris/EDTA
induce competence
addit of ice cold CaCl2 and heat shock at 42 to make bact envelope susceptible to DNA penetrat
methylase
catalyze addit of methyl groups to the recognition sequence so that the RE cannot cut the host's cell
alkaline lysis procedure
bact cell are pelleted and resuspended in a buffered medium

-lyse cells w/SDS, NaOH, RNAse
-S and R solub and denature prots
-NaOH have high pH to degrade RNA
alkaline condits-denature the host and foreign DNA separates their strands
-incr in salt conc by addit of potassium acetate cause the SDS, host chrom and prots to ppt while shorter plasmid renatures
spin column allows plasmids in SN to be purified by adsorption to a Si gel membrane that adsorbs DNA and leave RNA and prots
-plasmid is ppted w/EtOH and dissolved in Tris/EDTA buffer
-increase salt conc by addit of potass acetate which causes SDS, prots and host chrom to ppt

-SDS, prots, host chrom are centrifuged
-spin column allows plasmids in SN to be purified by adsorption to a Si gel membrane that adsorbs DNA and leaves RNA and proteins
I and III
restriction and methylase modificats in a single multisubunit complex, need ATP, cleave far
rec A mutants
does not recombine the foreign DNA with its own host chromosome since it lacks rec A proteins
phosphatase
removes phosphate and stops plasmid from forming bkbone
II
separate component for methylase activity, no ATP needed, and cleaves at the site, palindromic seqs
cohesive end
leave one strand of helix longer than complementary strand