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George Wolfe Loudoun County Public Schools Academy of Science [email protected] DNA Extraction and Amplification. EXTRACTION. ISOLATION OF DNA FROM INSECT SAMPLE ELIMINATION OF CELLULAR DEBRIS ELUTION OF PURIFIED DNA. STEP ONE OVERVIEW:ISOLATION.

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George Wolfe

Loudoun County Public Schools

Academy of Science

[email protected]


DNA Extraction and Amplification


EXTRACTION

  • ISOLATION OF DNA FROM INSECT SAMPLE

  • ELIMINATION OF CELLULAR DEBRIS

  • ELUTION OF PURIFIED DNA


STEP ONE OVERVIEW:ISOLATION

  • 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.


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.


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.


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!)


DNA AMPLIFICATION- PCR

  • Eluted or Control DNA (+ and -)

  • Master Mix

    • Taq Polymerase

    • Buffers

    • DNTP’s

    • MgCl2

  • Primers-Forward and Reverse (W spec)


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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