Beruflich Dokumente
Kultur Dokumente
TECHNIQUE
Objective
Students will model the process of
using restriction enzymes and
plasmids to form recombinant
DNA.
Background
Information
major tools of recombinant DNA
technology are bacterial enzymes
called restriction enzymes.
Enzyme cuts the backbones of the sticky ends of DNA fragments can be
molecules at that sequence. used to join DNA pieces originating
from different sources.
In order to be useful, the recombinant
DNA molecules have to be made to
replicate and function genetically
within a cell.
Blunt Ends
Sticky Ends
Expression vector
plasmid
Carries the gene of
interest in a specific
location that allows the
bacteria to express the
Plasmids gene
Contains:
Cloning Vector plasmid 1. Origin of
replication
2. Selectable
marker gene
3. Multiple
cloning site
Expression vector
plasmid
Contains
1. Origin of replication
2. Selectable marker
gene
3. An inducible
promoter
4. Transcription and
translation initiation
sequence
pARA-R plasmid
A sequence for initiating
DNA replication Regulates expression
of arabinose genes
Promoter site
Ampicillin resistant
Restriction digest of pARA-R
Biotech Experience
pARA-R
5,302 bp
PBAD-rfp
806 bp
Restriction analysis of pARA-R
Biotech Experience
4,496 bp
806 bp
Clone That Gene activity
RM2 and RM3
1. Cut the plasmid and the human
DNA with the appropriate
restriction enzyme
2. Insert the insulin gene into the
plasmid DNA
3. Determine which antibiotic you
would use to identify bacteria
that have taken in the plasmid
Plasmid DNA
Human DNA
Materials For each group:
Handout: Plasmid Base Sequence Strips
Scissors
Tape
Pencil
Paper
Instructions
1.Cut out the plasmid strips along the dotted lines.