1. Gel electrophoresis
The gel electrophoresis method was developed in the late 1960's. It is a fundamental tool for DNA sequencing.

2. + - How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
DNA + -
Power
small
large
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

3. Vertical Gel Electrophoresis for Fish
Remove comb
wells
Gel
Fill chamber with buffer
Break off

4. I.A. Calculate the Rf OF PROTEINS
1. Calculate the Rf value for each protein
band in the protein standard.
Rf = migration distance of protein (origin to band)
migration distance of dye front (origin to dye front)
Figure 2. Formula for calculating the retardation factor (Rf) of a protein in SDS-PAGE.

5. Ladder
Shark
Catfish
Salmon
Swordfish
Tuna
Flounder
Orange roughy

6. I.A. Rf OF PROTEINS
2. Plot the molecular weights of your standard proteins (vertical axis) versus the Rf values (horizontal axis) on semi-log graph paper.

7. 3. Draw a best-fit straight line through all your points.
I.A. Rf OF PROTEINS
3. Draw a best-fit straight line through all
your points.
Molecular wts.
Rf value

8. I.A. Rf OF PROTEINS
4. For each lane of fish protein extract, pick a single protein band and circle it on the graphic image that you will download from the class Laulima site. For each of these bands, determine its molecular weight using this standard curve.

9. I.B. PROTEIN FINGERPRINTING
Hypothesize Evolution Relationships Among the Fish Species Analyzed
I.B. PROTEIN FINGERPRINTING
Attempt to construct an evolutionary tree illustrating these relationships.

10. Loading the Gel
1. Lay out a small piece of Parafilm on a flat countertop.
2. Place a 1 μL drop of SYBR Gold dye (labeled "SG"; stains the DNA with a fluorescent dye) on the Parafilm.
3. Next to this drop, place a 1.7 μL drop of sample buffer (labeled "SB").
4. Next to these drops, place a 2.3 μL drop of sterile water.
5. Next to these drops place a 5 μL drop of your DNA sample and mix, using your pipetter all four drops together.
6. Take up the entire mixture (10 μL) into a pipetter and carefully transfer to one of the wells in the agarose gel. Be sure to
note which well your sample was applied to in your laboratory notebook.
Ladder:

11. Loading the Gel
7. Repeat these steps (1-6) with your PCR product, loading a different lane in the agarose gel.
8. The instructor will load a DNA ladder (allows molecular weight estimation of your DNA sample).
9. Once all of the wells are loaded, we will run the gel at 100 volts for about one hour.
10. After one hour has elapsed, we will visualize the gel under UV light.
11. Examine the gel image and identify which lanes contains your original DNA extract and your PCR product. Comparing the migration of your product to the DNA ladder, estimate the molecular weight of this product.

12. Cathode
(negative)
Anode
(positive)
DNA
buffer 
Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.

13. Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.
6X Loading Buffer: 
SYBR Gold Dye (for color)
Sample Buffer (SB)

14. Loading the Gel
Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

15. Running the Gel (red +) (black -)
Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.

17. Cathode
(-)
 wells
DNA
(-) 
 Bromophenol Blue
Gel
Anode
(+)
After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.

18. II.B. DNA Quantitation
Nanodrop spectrometer used to measure DNA concentrations

19. Nanodrop spectrometer
Clean with 1 μl nanopure water
Close, open
Wipe with kimwipe.
Blank
1 μl mastermix
Press blank button
Clean with kimwipe
MEASURE click left
Blank should read 0
Sample
1 μl DNA sample
READINGS
Concentration
Absorbance
260 nm
280 nm
Purity
Good purity if below 2 and above 1.8