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Plasmid DNA
Group # 5
Sim, Michelle D.
Suderio, Gellina Ann R.
Teope, Jonnah Kristina C.
Timbol, Danica Kaye P.
Uy, regina Celine DG
Plasmids
• First introduced by Joshua
Lederberg in 1952
• mostly circular double-stranded
DNA , few are linear, varies in
size
• extra-chromosomal DNA
molecule, capable of self
replicating
– replication is dependent on
host-cell proteins
• commonly used as cloning or
expression vectors
Types of Plasmids
(according to their ability to transfer to another bacteria)
• Conjugative Plasmids
– contain so-called tra-genes, which perform
conjugation
• Non-conjugative Plasmids
– incapable of initiating conjugation
– can only be transferred with the assistance of
conjugative plasmids
• Mobilizable Plasmids
– carry only a subset of the genes required for transfer
– can “parasitize” a conjugative plasmid
Types of Plasmids
(according to their function)
BamHI G•GATCC
EcoRI G•AATTC
HindIII A•AGCTT
PstI CTGCA•G
Types of Restriction Fragments
• Blunt-End Restriction Fragments
– are the result of restriction digestion which yields "blunt" or
"non-sticky" end DNA fragments
– the fragments do not have overhangs
– allows the cloning of incompatible DNA fragments
• Sticky-End Restriction Fragments
– are quite useful as they can be ligated with other compatible
restriction fragment ends (similar to the way lego pieces are
stuck together)
– allowed the cloning of DNA fragments into other DNA pieces
Restriction Enzyme Digestion
• employs the function of one or more restriction enzyme
to selectively cut DNA strands into shorter restriction
fragments
• Some restriction enzymes cut in the middle of their
recognition site, creating blunt-ended DNA fragments.
• However,the majority of enzymes make cuts staggered
on each strand, resulting in a few base pairs of single-
stranded DNA at each end of the fragment, known as
“sticky” ends.
• Some enzymes create 5' overhangs and others create
3‘ overhangs.
Restriction Enzyme Digestion
Experiment # 3
Materials Special Equipment
Plasmid pCH (0.2ug/uL) Microcentrifuge
Restriction enzymes
BamHI
EcoRI
HindIII
PstI
Restriction enzyme buffers
Micropipettors
0.5mL microcentrifuge tubes
Pipette tips
Crushed ice
Styrofoam cup
Procedure
Label 5 0.5mL microtubes (C B E H P)
C = no restriction enzyme
just uncut plasmid pCH
Place them in the microtube rack B = BamHI restriction digest
of plasmid pCH
E = EcoRI restriction digest
To each tube, add 7.5uL of distilled water, 1uL of
of plasmid pCH
plasmid pCH and 1uL of appropriate 10X restriction buffer
H = HindIII restriction digest
of plasmid pCH
To the appropriate labeled tubes, add 0.5uL of
C, B, E, H, and P P = PstI restriction digest
of plasmid pCH
BamHI 1 4361 bp
EcoRI 1 4361 bp
HindIII 1 4361 bp
PstI 1 4361 bp
Predict the number of fragments and their sizes that will be obtained when the
plasmid is cut by a combination of enzymes
HindIII + EcoRI 2
4359 – 29 = 4330 bp
4361 – 4330 = 31 bp
PstI + EcoRI 2
4359 – 3607 = 752
4361 – 752 = 3609