L20 Flashcards
whats the ouchterlony test
the plate with the holes that diffuse into each other
a vector is
a plasmid that has been streamlined and modified to make it amenable to carrying a payload of DNA
4 components that plasmids need
origin of replication
positive selection gene
inset differentiation gene
cloning sites
where do we insert foreign DNA on a plasmid
cloning site
how to make a positive selection gene
insert a gene for antibiotic resistance into the plasmid
what do viruses infect
bacteria
describe the restriction modification system
restriction is cutting
modification is the marks that mark that the DNA belongs to the cell
when does a restriction endonuclease cut
unmethylated palindrome sequences
what kind of enzyme is EcoR1
endonuclease
type 1 restriction enzyme
not a good tool
subunits for restriction and modification are the same and does not cut in a reproducible manner
type 2 restriction enzyme
good restriction enzyme
independent subunits and cuts in a reproducible manner
type 3 restriction enzyme
different subunits, but does not cut in a reproducible manner
best restriction enzyme type
type 2
will odd number sequences (palindrome sequences) be recognized and cut?
no - need an even number
what bp do restriction enzymes recognize
4, 6 or 8
specific condition needs for restriction enzymes (4)
temperature
Mg
pH
NaCl
what is always added last into the reaction
enzyme
whats a double digest reaction
reactions that use 2 enzymes at once if the reaction conditions are the same
sequential digest
1 reaction, then change the conditions, and add the second enzyme (2nd reaction) after denaturing the first
is DNA or protein bigger
DNA
agarose gel electrophoresis
used for DNA size separation
what color does DNA run to
red (anode)
where can ethidium bromide go when its combined with DNA to be visualized
in between the sets of base pairs
substitution to ethidium bromide
SyBr green
