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George Wolfe Loudoun County Public Schools Academy of Science GWOLFE1@LOUDOUN.GOV

George Wolfe Loudoun County Public Schools Academy of Science GWOLFE1@LOUDOUN.GOV. 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 GWOLFE1@LOUDOUN.GOV

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  1. George Wolfe Loudoun County Public Schools Academy of Science GWOLFE1@LOUDOUN.GOV

  2. DNA Extraction and Amplification

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

  4. 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.

  5. 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.

  6. 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.

  7. 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!)

  8. DNA AMPLIFICATION- PCR • Eluted or Control DNA (+ and -) • Master Mix • Taq Polymerase • Buffers • DNTP’s • MgCl2 • Primers-Forward and Reverse (W spec)

  9. 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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