1. Loudoun County Public Schools
George Wolfe
Loudoun County Public Schools
Academy of Science

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