Electrophoresis of DNA

1
Electrophoresis of DNA

• What is it used for?
• How does it work?
• How do we do it?
• What are the results?

2
What is electrophoresis?

• Separation of charged molecules
• DNA has net negative charge

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3
What is Agarose?

• A polysaccharide polymer
• Prepared from seaweed
• Highly purified
• Very water soluble
• Creates a gel
• Gel pore size depends on concentration of
  agarose

4
What is Agarose Gel Electrophoresis?
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• Larger DNA fragments migrate less
• Smaller DNA fragments migrate more

5
What is Agarose Gel Electrophoresis?
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• Larger DNA fragments migrate less
• Smaller DNA fragments migrate more

6
What is Agarose Gel Electrophoresis?
Agarose Gel Animation
7
Pouring a gel

• Calculate agarose amount (0.5-3)
• Measure agarose and add to buffer
• Melt in microwave
• Pour
• Let cool
• Remove comb

8
Preparing the gel for loading

• Place gel and tray into submarine chamber
• Fill with buffer

9
Preparing the DNA for loading

• DNA must be added to a loading dye
• Necessary because
• DNA must sink
• Need to track samples
• Loading dye contains
• Dye
• High density components (Ficoll, glycerol).

DNA (40 ul)
DYE (10 ul)
DNA Ready-to-Load (load 20 ul)
10
Loading a gel
Load underwater Let DNA flow from tip
11
Running a gel Part 1

• Connect to power supply
• Verify polarity!
• Voltage, time, current
• Run!

12
Running a gel Part 2

• Connect to power supply
• Verify polarity!
• Voltage, time, current
• Run!
• Stain with ethidium bromide or dye

Gel running
13
Analyzing the results

• Visualize bands (UV or visible light)
• Compare to standard
• Determine what DNA fragments are in your samples

14
Troubleshooting

• Bands run wrong direction
• Fuzzy bands
• Distorted bands
• Faint or no bands
• Bands crooked