Hold
micropipet
and
epitubes at
eye level
Micropipet Use
1 Add disposable pipet tip
2 Press plunger to first stop
3 Insert pipet tip into solution to be
transferred
4 Slowly release plunger to retrieve liquid
5 Move pipet tip into desired tube
6 Press plunger past first stop to second stop
to transfer liquid, keep the plunger down
as you remove it from the tube.
7 Eject tip
Micropipetting
technique A
technique B
Close the tips!
Micropipet
tip should
be ABOVE
the well
NOT IN
IT!!!!
Micropipet
tip punched
right
through the
gel
See dye
under the
wells
NICE!
When you to into the lab today, you
Each person in your group will transfer 15 uL of colored
will
firstmicrotube.
practice
the you
liquid into
an empty
Once using
this is completed,
should have 60 uL of brownish colored liquid in your practice
micropipette
tube.
15 uL
15 uL
15 uL
15 uL
After practicing how to use the micropipette, a
biotechnology student will demonstrate and
load the victim’s DNA into the gel.
You will then load your DNA into the gels.
Each student in the group can then load 1 well
of the gel with the suspect’s DNA.
There are four suspects total.
You may NOT load the gel without the help
of a biotechnology student watching you!
To understand electrophoresis, you
must understand the structure of
DNA
Phosphate groups of nucleotides have a - charge
5’
5’
2-
1
PO4
1
2
3’
OH
O
O-P=O
O
3
5’
4
2-
PO4
Phosphodiester bond
2
5
6
3’ OH
3’
Charge on a DNA Double Helix
3’
5’
1
2
3
4
5
Large groove
6
Small groove
7
8
9
10
1 Twist = 10.5 bp
5’
3’
Gel Electrophoresis
• Gel Electrophoresis: technique uses the
difference in electrical charge to separate
polymers (DNA, RNA, protein) on the basis
of size
Let’s see a model of how gel electrophoresis
works
DNA Electrophoresis analysis after DNA has
been cut by enzymes at various points, A and B
Restriction
enzymes
C
A
B A+B L
A
B
10 kb
A 8 kb
2 kb
B 7 kb
3 kb
A 5 kb
+ 3 kb
B 2 kb
Juang RH (2004) BCbasics
Purpose of Gel Electrophoresis?
• Get a “DNA Fingerprint” of individuals
• Trace a sequence of genetic markers in
families
• Diagnose disease
• Compare genes of different people,
different species
• Find mutations
• Paternity tests
Restriction Enzymes
•
•
•
•
•
•
•
rDNA (recombinant DNA)—the produced piece of DNA from inserted
another piece of DNA
recognize specific sites to cut the DNA
Blunt ends—straight across
Sticky ends—one side of DNA is longer than the other, these overhangs allow
for complementary matches between two DNA pieces cut by the same
enzyme, the sticky ends match and pasting ma occur to produce an rDNA
molecule
More than 1200 restriction enzymes discovered & isolated from bacteria
Read 5 3
Palindromic (example radar or
GAATTC
CTTAAG
Reviewing Homework
Based on this DNA
Fingerprint created,
who should the
police arrest as the
prime suspect?
Suspect 1 or 2?
Why?