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DNA Extraction and Amplification

DNA Extraction and Amplification. EXTRACTION. ISOLATION OF DNA FROM INSECT SAMPLE ELIMINATION OF CELLULAR DEBRIS ELUTION OF PURIFIED DNA. STEP ONE OVERVIEW:ISOLATION. Maceration with PBS –exposes cell components without molecular degradation Cell Lysis Solution-destroys cell membranes

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DNA Extraction and Amplification

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  1. DNA Extraction and Amplification

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

  3. STEP ONE OVERVIEW:ISOLATION • Maceration with PBS –exposes cell components without molecular degradation • Cell Lysis Solution-destroys cell membranes • Proteinase K-Destroys DNAses At this point you have a “soup” of cellular components. The DNA must now be removed.

  4. 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. Think about the numbers….This means we will do 5 separate extractions…3 unknowns, 1 positive control, 1 negative control.

  5. 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.

  6. Some Details • Use of Controls • Optional Stopping Points • After elution and centrifugation the DNA will be invisible…Trust!!!

  7. Places where your students (but certainly not you) will mess this up • Losing track of what you have or have not added. • 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!) • Mixing waste with eluted DNA. • Bad pipetting 

  8. DNA AMPLIFICATION- PCR • Eluted and Control DNA (+ and -) from the insects (5 tubes) • We will be adding 1 additional prep…an already eluted + DNA sample. • Master Mix (all incorporated into a bead) • 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. (yet again) • More bad pipetting.

  10. Keeping Your Students Organized

  11. Keeping Track of the Steps

  12. George Wolfe Loudoun County Academy of Science Sterling, Va. 20165 george.wolfe@loudoun.k12.va.us

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