Download + Agarose gel

Last class
Class policies,
etc.
VNTRs
PCR
Visualizing DNA
DNA is not colored; Can’t see
in solution.
Separate pieces based on
____________
Run mixture through an
__________ gel
Agarose solution is like JellO.
Liquid when hot, solid(ish)
when cold
- Agarose is NOT edible
- Agarose tastes really,
really bad
Separating DNA
Agarose gel is porous
Small pores in gel allow DNA to pass
through
_______ DNA passes easily, _____
DNA less easily
Agarose gel
-
+
Agarose gel
http://www.youtube.com/watch?v=2UQIoYhOowM
Loading dye
Why do you need a “buffer?”
Why do you add glycerol?
Why do you need the “dye”?
Seeing the DNA
Gel/buffer contains
ETHIDIUM BROMIDE
(EtBr)
EtBr + UV light =
________
fluorescence
EtBr in DNA + UV
light = __________
fluorescence
Regions in gel with
DNA will show a
BRIGHT “band”
http://wikidoc.org/index.php/Agarose_gel_electrophoresis
Estimating size of DNA
See DNA. But size?
Run DNA fragments of known
length
Distance run = size of DNA
(compare to ladder)
2000 bp
1000 bp
600 bp
500 bp
400 bp
300 bp
200 bp
http://wikidoc.org/index.php/Agarose_gel_electrophoresis
Changing gel parameters
Wh
y?
Increase voltage
Increase percentage
of gel
Increase time of run
Watch
out!
LAB 2
Gel will be poured for you
Load and run gel
Check results
Lab 3 onwards = pour
your own gel
How do we study life?
I think “X” BECAUSE “Y”
Design an experiment to PROVE
“X”
Experiment: Predict outcomes if
“X” is TRUE
Predict outcomes if “X” is
Do
experiment, observe results
FALSE
Results; Therefore “X” is
TRUE/FALSE
Unexpected results/observations =
Restriction endonucleases
RE is an enzyme!
- Optimal temperature
- Optimal salt
concentration/composition
- Optimal pH
- Time of activity
RE cutting is random!
Designing experiments
Tube
10X Buffer
RE (µL)
(µL)
Water
1N HCl (µL)
(µL)
1
1.0
1.0
-
8.0
0.0
2
1.0
1.0
1.0
7.0
0.0
3
1.0
1.0
-
7.0
1.0
4
1.0
1.0
1.0
6.0
1.0
5
1.0
1.0
-
6.0
2.0
6
1.0
1.0
1.0
5.0
2.0
Designing experiments
Tube
DNA (µl)
10X Buffer
RE (µL)
(µL)
Water
1N HCl (µL)
(µL)
1
1.0
1.0
-
8.0
0.0
2
1.0
1.0
1.0
7.0
0.0
3
1.0
1.0
-
7.0
1.0
4
1.0
1.0
1.0
6.0
1.0
5
1.0
1.0
-
6.0
2.0
6
1.0
1.0
1.0
5.0
2.0
What variable is being
tested?
Draw
What are
controls?
Additional
controls?
Designing experiments
Form a
hypothesis
Design an
experiment
Predictions?
LAB 2
Design experiment to test
ONE variable
- Time
- Buffer
- Enzyme concentration
Discuss with partner, other
groups & TA
Generate experimental design
Include controls!
Interpreting results: Making predictions
Effect of variable on RE digestion of
linear 10Kb DNA
Complete digestion with RE = 2,3, 5Kb
bands
Draw gel for 0%, 10%, 50% and 75%
digestion