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Unformatted text preview: Restric(on Enzymes aka __________________ What are they/What do they do? • Enzymes that recognize/bind and cleave speciﬁc 4‐8 bp sequences in dsDNA, called “restric'on sites” • crea(ng reproducible restric'on fragments • Most restric(on sites are short inverted sequences (palindromes!) Usually dimers that recognize both strands Bacteria Structure Naturally occurring in what organism? Na(ve func(on/purpose? How does bacteria protect its own DNA? Ways they cut (3) Common ones Prac(ce Problems 1. 2. Restric(on enzyme X recognizes a 5bp sequence, how frequently will it cut? about every ______ bps What are 2 ways to achieve less frequent cu[ng of DNA (to get larger fragments)? 3. Construct a restric(on map of a linear fragment of DNA, using the following data. Your map should indicate the rela(ve posi(ons of the restric(on sites along with distances from the ends of the molecule to the restric(on sites and between restric(on sites: DNA uncut DNA 10,000 DNA cut w/ EcoRI DNA cut w/ BamHI DNA cut w/EcoRI + BamHI Sizes of Fragments (bp) 8000, 2000 5000, 5000 5000, 3000, 2000 4. Shown below is a ﬁc((ous 4,000bp plasmid. Create a ﬁgure predic(ng the appearance of a gel showing digests with EcoRI, BamHI, and EcoRI + BamHI combined. Include a lane showing a ladder for size reference. A new plasmid DNA has been isolated from E.coli. It was found to have a single site for the restric(on enzyme SmaI. Explain how you can use electron microscopy to determine whether the plasmid replicates unidirec(onally or bidirec(onally. Bacteria Genome Plasmid Eukaryotes λ phage outside host Inside host shape+make‐up Mode of replica(on #replicons #forks/replicon Rolling circle ‐‐‐‐‐ ‐‐‐‐‐‐ Name and BRIEFLY describe (3 sentences MAX) 4 diﬀerent proteins involved in the pre‐inita'on of DNA replica(on at an E.coli Ori (PRIOR to DNA polymerase III). (a) (b) (c) (d) In the Meselson & Stahl experiment E.coli cells were transferred from 15N containing heavy medium into 14N containing light medium and samples were removed at various (mes for analysis of the DNA by CsCl equilibrium centrifuga(on. What frac'on of the DNA would be expected at light, hybrid and heavy densi(es a=er 3 doublings based on the following models of replica(on: (A) Semi‐conserva've Light _________ Hybrid ________ Heavy ________ (B) Conserva've Light _________ Hybrid ________ Heavy ________ ...
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- Spring '08