Download Plasmid w/ kanamycin resistance (pKAN)

Stage 1: Prepare your plasmids to be
cut by restriction enzymes
• Obtain the plasmids (pKAN and pAMP)
P stands for plasmid
pKAN = plasmid with antibiotic kanamycin resistance
pAMP = plasmid with antibiotic ampicillin resistance
• Mix plasmids with restriction enzymes
– BamH1 and Hind III
– Restriction enzymes cut the plasmids at precise
locations
Plasmid w/ kanamycin resistance
(pKAN)
Restriction enzyme:
Hind III
(cuts @ bp 234)
pKAN = 4194 bp
Restriction enzyme:
Bam HI
(cuts @ bp 2095)
2333 bp
restriction
fragment
1861 bp
restriction
fragment
Thousands of plasmids in our samples
K+
(digested plasmid)
K–
(uncut plasmid)
+ means the restriction enzymes were
added
- means the restriction enzymes not
added
How many plasmid fragments?
How many plasmid fragments?
Stage 2: Check to see if the restriction
enzymes worked
• DNA electrophoresis
– Plasmid fragments are loaded into a gel
– Connected to a power supply
– Separates fragments based on their sizes
– Smaller fragments travel further through the gel
We will then micropipette the plasmids
Electrophoresis: sizes DNA fragments
Load the plasmids into an electrophoresis chamber
Connect the electrophoresis to a power
supply…DNA has a negative electric charge.
10,000bp
A-
A+
K-
K+
Known DNA
markers
Electrophoresis:
sizes DNA
fragments
How many
marks will
appear in the Kand A- lanes?
8,000bp
6,000bp
5,000bp
4,000bp
3,000bp
2,000bp
Look at the lab
handout and
let’s predict the
A+ fragments
1,500bp
1,000bp
500bp
What just
happened?
Why not just 1
band?
8,000bp
6,000bp
5,000bp
4,000bp
3,000bp
2,000bp
1,500bp
1,000bp
500bp
Marker DNA
A-
K-
A+
K+
A+
K+
A+
K+
A+
K+
Marker DNA
10,000bp
2008 Data/Results
So now what???
• Plasmids have been
engineered for
human uses. The
human gene for
insulin (red) can now
be added to the
plasmid. The
bacteria will produce
insulin for diabetics!
So now what???
The bacteria with
the recombinant
DNA replicates,
thus passing the
insulin gene onto
its offspring. Each
cell now will
produce insulin
for humans to
harvest and use.
8,000bp
6,000bp
5,000bp
4,000bp
3,000bp
2,000bp
1,500bp
1,000bp
500bp
Marker DNA
A-
K-
A+
K+
A+
K+
A+
K+
A+
K+
Marker DNA
10,000bp
8,000bp
6,000bp
5,000bp
4,000bp
3,000bp
2,000bp
1,500bp
1,000bp
500bp
Marker DNA
A-
K-
A+
K+
A+
K+
A+
K+
A+
K+
Marker DNA
10,000bp