1. DNA Extraction and Amplification

2. EXTRACTION 1. ISOLATION OF DNA FROM INSECT SAMPLE 2. ELIMINATION OF CELLULAR DEBRIS 3. ELUTION OF PURIFIED DNA

3. STEP ONE OVERVIEW:ISOLATION 1. Maceration with PBS –exposes cell components without molecular degradation 2. Cell Lysis Solution-destroys cell membranes 3. 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 1. Cell “soup” is added to a spin column. 2. A filter in the column attracts DNA, Proteins, and other cell components. 3. 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 1. The DNA is still stuck to the original filter in the spin column. 2. Everything else should be gone. 3. A final Elution Buffer is added, the sample is centrifuged, this removes the DNA.

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

7. Places where your students (but certainly not you) will mess this up Places where your students (but certainly not you) will mess this up 1. Losing track of what you have or have not added. 2. Not labeling tubes properly. 3. Waiting too long to add Proteinase K*** 4. Not changing pipette tips and macerators. 5. Throwing out their eluted DNA (yes, this is a common mistake!) 6. Mixing waste with eluted DNA. 7. Bad pipetting 

8. DNA AMPLIFICATION- PCR 1. Eluted and Control DNA (+ and -) from the insects (5 tubes) 2. We will be adding 1 additional prep…an already eluted + DNA sample. 3. Master Mix (all incorporated into a bead) Taq Polymerase Taq Polymerase Buffers Buffers DNTP’s DNTP’s MgCl 2 MgCl 2 4. Primers-Forward and Reverse (W spec)

9. More places for your students (but not you) to mess up! 1. Tube Labeling 2. Keeping track of what has been added. (yet again) 3. 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