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Plasmids and Vectors
Plasmids and Vectors
Instructor Supplement to
pGlo Bacterial Transformation
Antibiotic
Resistance
Gene
Multiple
Cloning
Site
Asilomar Conference
People
NIH
It
That
NIH Guidelines
Contents
Subsequent modifications
efficiency of
transformation
Easier to restriction map
Higher copy numbers
pBR322
Clone fragment in one
antibiotic gene
Select for other antibiotic
resistance
AmpR
Screen for presence of
one resistance gene
(selects against
untransformed bacteria) APstI
and loss of resistance to
interrupted antibiotic
resistance gene (selects
for recombinant
molecule)
EcoRI
TetR
pBR322
4,361 bp
BamHI
The inclusion of
polylinkers into
plasmid vectors
Polylinker is a tandem
array of restriction
endonuclease sites in a
very short expanse of
DNA
For example, pUC18s
polylinker
Sites for 13 REs
Region spans the
equivalent of 20
amino acids or 60
nucleotides
Bacteriophage lambda ()
A virus that infects
bacteria
o In 1971 Alan
Campbell showed
that the central third
of the genome was
not required for lytic
growth. People
started to replace it
with E. coli DNA
o
Lambda genome is
approximately 49
kb in length.
Only 30 kb is
required for lytic
growth.
Thus, one could
clone 19 kb of
foreign DNA.
Packaging
efficiency 78%100% of the
lambda genome.
A complete animation of the lytic cycle:
http://www.blackwellpublishing.com/trun/artwork/Animations/Lambda/lambda.html
Bacteriophage lambda
Protein capsule of
COS site: Cohesive
lambda has a tight
sticky ends
constraint on the
Lysis
Head
amount of DNA
Replication
that will fit inside
ori
it (~ 55kb)
Tail
By the early
Circularized
1970s we knew
lambda
that a good
Lysogeny
portion of lambda
was not required
Junk DNA
Eliminate the
non-essential
parts of lambda
Can now insert
large pieces of
DNA (~ 20 kb)
Lysis
Replication
ori
COS
Head
Tail
But:
Cosmids
ori
TetR
21.5 kb
cos
EcoRI
Cos site is the only
requirement for
packaging into
phage particle
Other Vectors
Genomic Library
25
cDNA library
Screening libraries
by colony hybridization
Restriction Mapping