Cell Bio Lab

Front 1

sanger method

Back 1

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'

Front 2

spin column

Back 2

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

Front 3

induce competence

Back 3

addit of ice cold CaCl2 and heat shock at 42 to make bact envelope susceptible to DNA penetrat

Front 4

methylase

Back 4

catalyze addit of methyl groups to the recognition sequence so that the RE cannot cut the host's cell

Front 5

alkaline lysis procedure

Back 5

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

Front 6

I and III

Back 6

restriction and methylase modificats in a single multisubunit complex, need ATP, cleave far

Front 7

rec A mutants

Back 7

does not recombine the foreign DNA with its own host chromosome since it lacks rec A proteins

Front 8

phosphatase

Back 8

removes phosphate and stops plasmid from forming bkbone

Front 9

II

Back 9

separate component for methylase activity, no ATP needed, and cleaves at the site, palindromic seqs

Front 10

cohesive end

Back 10

leave one strand of helix longer than complementary strand