•Verify recombination by
electrophoresis.
•Digest of rfp gene.
•Transform bacteria with
recombinant plasmid.
•Recombination (ligation) of
plasmid and rfp gene.
•Induce expression of rfp gene.
•Observe bacteria.
•Digest of plasmid
Journal 12.02.11
1) Copy the steps on the other
slide in the correct order.
2) Flip the pages in the book:
which steps correspond to
labs 2a, 4a, 5a?
3) At which step is there a
process of gene expression?
(Can be answered in 3 columns)
DO: Lab 2a – Restriction digest
1. Mix reagents, as described in
page 2a.3
-Reaction buffer mix
-Plasmid
-Water (to one test tube)
-Add enzyme: from teacher.
2. Place at 37oC for at least 1
hour.
•PreLab 2a – Q & A
• Animation: Plasmid digestion
Group papers:
•Lab 1 – Conclusions page 1.6
•Lab 2a – Conclusions page 2a.4
-Pour Gel (not in 2013)
http://Plasmid
Recombination
Gene Cloning Animation
pARA-R construct
Recombinant plasmid of interest
pARA-R
4720 bp
rfp
702bp
rfp – Gene for Red Fluorescent
Protein, originally from the Sea
Anemone Discosoma sp.
To presenting students:
The following slide is from the
alternative lab sequence, where
students also performed the
ligation step.
The two plasmids: One served as
the ‘source’ of the rfp gene, and
one as the ‘vector’ which would
turn into our pARA-R.
Restriction analysis of pKAN-R and pARA
Wallace
pKAN-R
BamHI
5,512 bp
pARA
P -rfp
806 bp
4,872 bp
BAD
376 bp
Restriction analysis of pKAN-R and pARA
Wallace
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4,706 bp
BamH I
Hind III
4,496 bp
Hind III
BamH I
806 bp
Hind III
BamH I
376 bp
Engineering the Plasmid: ligation of rfp gene into p-ARA
BamH I
sticky end
Hind III
sticky end
Hind III
sticky end
BamH I
sticky end
Restriction digest of pARA-R
Recombinant plasmid of interest
pARA-R
4720 bp
rfp
702bp
To presenting students:
Refer to Lab3 (background and
conclusions) in the student guide
for more information.
Key points:
-Why 70oC incubation
(Denaturation – what does this
mean?)
- What does ligation have to do
with ‘recombinant DNA’?