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Ch. 2A: How Do You Begin
to Clone a Gene?
Learning goals
Describe the characteristics of plasmids
Explain how plasmids are used in cloning a
gene
Describe the function of restriction enzymes
Explain how to use restriction enzymes to
create a recombinant plasmid
Key Ideas
Plasmids - ideal vectors for genetic engineering
–
–
–
–
replicate in the bacteria cell
gene promoter
antibiotic resistance as a selectable marker,
can be transferred into bacteria by conjugation.
Restriction enzymes – key to the creation of a
recombinant plasmid.
– cut DNA at specific sequences
– sticky ends allow strands to join
The Plasmid
pARA-R plasmid
pARA-R construct
Bruce Wallace
Recombinant plasmid of interest
pARA-R
5,302 bp
PBAD-rfp
806 bp
Engineering the Plasmid: ligation of rfp gene into p-ARA
Bruce Wallace
BamH I
sticky end
3’
5’
3’
5’
sticky end
5’
5’
3’
3’
Hind III
BamH I
sticky end
Hind III
sticky end
3’
5’
3’
5’
Restriction digest of pARA-R
Biotech Experience
Recombinant plasmid of interest
pARA-R
5,302 bp
PBAD-rfp
806 bp
Restriction analysis of pARA-R
Biotech Experience
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4,496 bp
Hind III
BamH I
806 bp
Learning goals: lab #4
Describe why it is important to verify products
created in the genetic engineering process
Predict the relative speed of DNA restriction
fragments and plasmids through a gel during
gel electrophoresis
Separate and identify DNA restriction fragments
and plasmids using gel electrophoresis
Key Ideas
The multistep process that is used to clone a
gene results in multiple products
– Need to verify that you have the recombinant
plasmid you need.
DNA fragments and plasmids can be separated
by gel electrophoresis.
Loading dye helps monitor the progress of the
gel electrophoresis procedure.
DNA ladder helps determine the sizes of
unknown pieces of DNA
Gel is stained in order to show the location of the
DNA fragments and plasmids.
Review questions
1. Why is it important to have sticky ends?
2. What is the purpose of the restriction
enzymes?
3. How do you confirm the uptake of the
gene into the plasmid?
Clone That Gene
activity
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
Tips
Reagents should be stored in a freezer until you
are ready to prepare them for students. Allow
to defrost for 15 minutes before using.
The reagents can be aliquoted up to several days
before the lab, then store in the
freezer/refrigerator
Vortex and spin enzyme mix and 2.5x RB before
aliquoting
video
Tips
Low-tech water bath
Calibrate and mark the controller
Multiple pans allow tubes from
different classes to stay separated
Bottom of tubes in the water
“Floatie” marked with team
number. Each period has a
different color.