George Wolfe

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• George Wolfe
• Loudoun County Public Schools
• Academy of Science
• GWOLFE1_at_LOUDOUN.GOV

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DNA Extraction and Amplification
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EXTRACTION

• ISOLATION OF DNA FROM INSECT SAMPLE
• ELIMINATION OF CELLULAR DEBRIS
• ELUTION OF PURIFIED DNA

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STEP ONE OVERVIEWISOLATION

• Cell Lysis Solution-destroys cell membranes
• Proteinase K-Destroys DNAses
• Protein Precipitating Solution precipitates
  Protein (duh)!
• At this point you have a soup of cellular
  components. The DNA must now be removed.

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STEP TWO-ELIMINATION OF CELLULAR DEBRIS

• Cell soup is added to a spin column.
• A filter in the column attracts DNA, Proteins,
  and other cell components.
• A series of buffers/centrifugations will wash out
  everything but the DNA.

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Step 3-DNA Elution

• The DNA is still stuck to the original filter in
  the spin column.
• Everything else should be gone.
• A final Elution Buffer is added, the sample is
  centrifuged, this removes the DNA.

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Places where your students (but certainly not
you) will mess this up?

• Losing track of what you have or have not added
  (see organization chart)
• Not labeling tubes properly.
• Waiting too long to add Proteinase K
• Not changing pipette tips and macerators
• Throwing out their eluted DNA (yes, this is a
  common mistake!)

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DNA AMPLIFICATION- PCR

• Eluted or Control DNA ( and -)
• Master Mix
• Taq Polymerase
• Buffers
• DNTPs
• MgCl2
• Primers-Forward and Reverse (W spec)

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More places for your students (but not you) to
mess up!

• Tube Labeling
• Keeping track of what has been added.
• We are using pelleted master mix
• See organizational chart

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