Lab 5 Flashcards
Restriction enzyme/endonuclease
produced by bacteria as a
defense mechanism to digest the DNA of foreign invaders such as bacterial viruses.
What do restriction enzymes/endonucleases do?
an enzyme that cuts
double-stranded or single stranded DNA at specific recognition nucleotide sequences
known as restriction sites.
Restriction
Inside a
bacterial host, the restriction enzymes selectively cut up foreign DNA
modification enzyme
(a methylase) to protect it
from the restriction enzyme’s activity
To cut the DNA, a restriction enzyme …
makes two
incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA
double helix.
recognition sequence/site
of
any DNA-binding protein motif that exhibits binding specificity, refers to the DNA
sequence (or subset thereof), to which the domain is specific.
Most restricti
inverted repeat
the sequence of the top strand of DNA is
identical to that of the bottom strand read in the opposite direction.
restriction endonuclease
an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites
recognition sequence
a DNA sequence to which a structural motif of a DNA-binding domain exhibits binding specificity
blunt end
The simplest DNA end of a double stranded molecule is called a blunt end. Blunt ends are also known as non-cohesive ends. In a blunt-ended molecule, both strands terminate in a base pair
sticky end
the ends left have one strand overhanging the other to form a short (typically 4 nt) single-stranded segment. This overhang will easily re-attach to other ends like it
overhanding sing-strand
a stretch of unpaired nucleotides in the end of a DNA molecule
alkaline phosphatase
a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation.
antarctic phosphatase
a heat labile alkaline phosphatase purified from a recombinant source. AnP nonspecifically catalyzes the dephosphorylation of 5´ and 3´ ends of DNA and RNA phosphomonoesters.
procedure
enzymes on ice
spin down enzymes before use
add NEB, BSA, distilled water, plasmid, and BamH1 enzyme together (50 uL total)
mix the sample with a 30 second spin
Incubate tubes at 37 degrees C in the water bath for 3 hours or overnight
