Midterm Prep Flashcards
(55 cards)
Name 3 characteristics of restriction enzymes type I
cleave at sites remote from recognition site;
require both ATP and S-adenosyl-L-methionine to function;
multifunctional protein with both restriction and methylase activities
Name 3 characteristics of restriction enzymes type II
cleave within or at short specific distances from recognition site;
most require magnesium;
single function (restriction) enzymes independent of methylase
Name 3 characteristics of restriction enzymes type III
cleave at sites a short distance from recognition site; require ATP (but do not hydrolyse it);
S-adenosyl-L-methionine stimulates reaction but is not required;
exist as part of a complex with a modification methylase
First step in DNA synthesis
unwinding (separating?)
What is M13?
M13 is a filamentous bacteriophage composed of circular single stranded DNA (ssDNA) which is 6407 nucleotides long
Alt answer
M13 (or f1) phages are filamentous phages that infect E. coli through pili, and are able to produce new virions without lysing the host cell. M13 has some key structural elements:
Circular, single-stranded DNA
~6.4 kb long
10 genes in the genome.
Enterobacteria phage λ (lambda phage, coliphage λ):
is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli (E. coli).
isoschizomers
restriction endonucleases that recognize the same sequence
prototype
The first example of isoschizomer discovered is called a prototype and all subsequent enzymes that recognize the same sequence are isoschizomers of the prototype.
neoschizomers
neoschizomers are a subset of isoschizomers that recognize the same sequence, but cleave at different positions from the prototype.
Unit:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C (Temp. varies as per the enzyme taken) in a total reaction volume of 50 µl
Store DNA at what temp?
Short-term storage (weeks) at 4°C in Tris-EDTA
Medium-term storage (months) at –80°C in Tris-EDTA
Long-term storage (years) at as –80°C as a precipitate under ethanol
Long-terms storage (decades) at –164°C or dried
In what direction is DNA read?
All known DNA replication systems require a free 3’ hydroxyl group before synthesis can be initiated (Important note: DNA is read in 3’ to 5’ direction whereas
In what direction is DNA synthesized?
a new strand is synthesized in the 5’ to 3’ direction
In what direction is mRNA synthesized?
a new strand is synthesized in the 5’ to 3’ direction
In what direction is mRNAs read?
All mRNAs are read in the 5´ to 3´ direction, and polypeptide chains are synthesized from the amino to the carboxy terminus.
Plasmids:
self-replicating
extra chromosomal
DNA
in virtually all bacterial species
fusion proteins:
chimeric proteins (literally, made of parts from different sources) are proteins created through the joining of two or more genes that originally coded for separate proteins.
what is equation to give expected ng of insert amount?
(ng vector X kb size insert/ kb size vector) X molar ratio
what are the seven steps of cloning?
1) select vector
2) select insert
3) cut V&I
4) ligate V&I
5) transform cells
6) screen cells
7) amplify cells
What wavelength do NA have absorbance maxima?
260 nm
What ratio indicates purity of NA?
260:280
What are two types of plasmids?
Cloning vectors and expression vectors
Define vector:
a DNA molecule (often a plasmid) capable of replication in a host organism, specially modified for propagating DNA sequences
Define recombinant DNA:
DNA created in a test tube by ligating together pieces of DNA that are not normally contiguous
