DNA Extraction DNA Extraction and Amplificationand Amplification

EXTRACTIONEXTRACTION1.1. ISOLATION OF DNA ISOLATION OF DNA

FROM INSECT SAMPLEFROM INSECT SAMPLE

2.2. ELIMINATION OF ELIMINATION OF CELLULAR DEBRISCELLULAR DEBRIS

3.3. ELUTION OF PURIFIED ELUTION OF PURIFIED DNADNA

STEP ONE STEP ONE OVERVIEW:ISOLATIONOVERVIEW:ISOLATION

1.1. Maceration with PBS –exposes cell Maceration with PBS –exposes cell components without molecular components without molecular degradationdegradation

2.2. Cell Lysis Solution-destroys cell Cell Lysis Solution-destroys cell membranes membranes

3.3. Proteinase K-Destroys DNAsesProteinase K-Destroys DNAsesAt this point you have a “soup” of At this point you have a “soup” of cellular components. The DNA must now cellular components. The DNA must now be removed.be removed.

STEP TWO-ELIMINATION STEP TWO-ELIMINATION OF CELLULAR DEBRISOF CELLULAR DEBRIS

1.1. Cell “soup” is added to a spin column.Cell “soup” is added to a spin column.2.2. A filter in the column attracts DNA, A filter in the column attracts DNA,

Proteins, and other cell components.Proteins, and other cell components.3.3. A series of buffers/centrifugations will A series of buffers/centrifugations will

wash out everything but the DNA.wash out everything but the DNA.Think about the numbers….This means Think about the numbers….This means

we will do 5 separate extractions…3 we will do 5 separate extractions…3 unknowns, 1 positive control, 1 unknowns, 1 positive control, 1 negative control.negative control.

Step 3-DNA ElutionStep 3-DNA Elution

1.1. The DNA is still stuck to the The DNA is still stuck to the original filter in the spin column.original filter in the spin column.

2.2. Everything else should be gone.Everything else should be gone.

3.3. A final Elution Buffer is added, A final Elution Buffer is added, the sample is centrifuged, this the sample is centrifuged, this removes the DNA. removes the DNA.

Some DetailsSome Details

Use of ControlsUse of Controls Optional Stopping PointsOptional Stopping Points After elution and centrifugation the After elution and centrifugation the

DNA will be invisible…Trust!!!DNA will be invisible…Trust!!!

Places where your students Places where your students (but certainly not you) will (but certainly not you) will

mess this upmess this up1.1. Losing track of what you have or have Losing track of what you have or have

not added.not added.2.2. Not labeling tubes properly.Not labeling tubes properly.3.3. Waiting too long to add Proteinase Waiting too long to add Proteinase

K***K***4.4. Not changing pipette tips and Not changing pipette tips and

macerators.macerators.5.5. Throwing out their eluted DNA (yes, Throwing out their eluted DNA (yes,

this is a common mistake!)this is a common mistake!)6.6. Mixing waste with eluted DNA.Mixing waste with eluted DNA.7.7. Bad pipetting Bad pipetting

DNA AMPLIFICATION- DNA AMPLIFICATION- PCRPCR

1.1. Eluted and Control DNA (+ and -) Eluted and Control DNA (+ and -) from the insects (5 tubes)from the insects (5 tubes)

2.2. We will be adding 1 additional We will be adding 1 additional prep…an already eluted + DNA prep…an already eluted + DNA sample.sample.

3.3. Master Mix (all incorporated into a Master Mix (all incorporated into a bead)bead)

Taq PolymeraseTaq Polymerase BuffersBuffers DNTP’s DNTP’s MgClMgCl22

4.4. Primers-Forward and Reverse (W spec)Primers-Forward and Reverse (W spec)

More places for your More places for your students (but not you) to students (but not you) to

mess up!mess up!1.1. Tube LabelingTube Labeling

2.2. Keeping track of what has Keeping track of what has been added. (yet again)been added. (yet again)

3.3. More bad pipetting.More bad pipetting.

Keeping Your Students Keeping Your Students OrganizedOrganized

Keeping Track of the Keeping Track of the StepsSteps

George WolfeGeorge Wolfe

Loudoun County Academy of ScienceLoudoun County Academy of Science

Sterling, Va. 20165Sterling, Va. 20165

george.wolfe@loudoun.k12.va.usgeorge.wolfe@loudoun.k12.va.us