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The following procedures were used to isolate DNA from 10 species of Drosophila at the Woodrow Wilson Fellowship Foundation's CORE Institute, Teaching in the Genome Age, July 7 - August 3, 2002.

Our Procedures

DNA Isolation
Polymerase Chain Reaction
Visualizing DNA (Electrophoresis)
Gene Cleaning
Sequencing Reaction
Assessing the Sequence (BLAST)

DNA  Isolation

Purpose: The purpose of DNA isolation is to obtain a fairly clean sample of a specimens DNA, that can then be used for additional studies.

Materials: flies, microcentrifuge tubes, micropestle, 100% EtOH, DNeasy Kit (ATL buffer,  Proteinase K, AL buffer, spin column and collection tube, AW1 buffer, AW2 buffer, and AE buffer)

Procedure:

1. Place 2 - 5 flies in a 1.5 mL microcentrifuge tube.  Add 180 µL ATL (Tissue Lysis) buffer, using micropestle grind flies beyond recognition (this means no recognizable parts, except for the wings).

   Fly masher at work!

2. Add 20 µL Proteinase K, VORTEX, incubate at 55°C for 1 - 3 hours.  Prepare 2 - 1.5 mL microcentrifuge tubes for each isolation, label with date, taxon, and elution # (1 or 2).
3. Retrieve the samples and VORTEX for 15 seconds.  Add 200 µL AL (Lysis) buffer (to lyse cells), VORTEX, incubate in the heating block at 70°C for 10 minutes.
4. Add 200 µL 100% EtOH, VORTEX.  Pipette fly mixture (~750 µL) into spin column in collection tube.
5. Spin at 8000 rpm for 1 minute, discard collection tube and filtrate.
6. Place spin column into fresh collection tube.  Add 500 µL AW1 (Wash 1) buffer.
7. Spin at 8000 rpm for 1 minute, discard collection tube and filtrate.
8. Place spin column into fresh collection tube.  Add 500 µL AW2 (Wash 2) buffer.
9. Spin at FULL SPEED for 3 minutes, discard collection tube and filtrate.
10. Place spin column into your prepared 1.5 mL microcentrifuge tube marked "elution 1."  Add 200 µL AE (Elution) buffer.  Incubate at room temperature for 1 minute.
11. Spin at 8000 rpm for 1 minute, this is your DNA.
12. Repeat steps 10 and 11 for "elution 2" (This elution will be less concentrated, but usable.)
13. Store in freezer (-20°C) for long term storage.  A small aliquot can be stored 2 - 3 days at 4°C.

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Polymerase Chain Reaction (PCR)

Purpose: PCR is a laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large sample of a piece of DNA that can later be analyzed.

Visual Link: Look at the following site to see an animated, Science2Discover at http://www.science2discover.com/.  Additional information on PCR can be found at http://www.dnalc.org/shockwave/pcranwhole.html.

Materials: working stock, H₂O (dna & dnase free), primer (gls forward, reverse and sia forward, reverse), PCR tube

Procedure:

1. Obtain materials, label PCR tubes
2. Add the following to each PCR tubes.  See Table A.
3. Run the sample through the following PCR cycle, program the machine.  See Table B.

   The PCR Machine

4. Run the sample on a gel to see the PCR product.

Table A

Table B

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Visualizing DNA (Electrophoresis)

Purpose: DNA electrophoresis was done to visualize the PCR product to ensure that PCR product was made.

Materials: PCR product, E-gels, loading dye

Procedure:

1. Add 7 µL of PCR product to separate tubes.
2. Add 1µL of loading dye.
3. Load the sample into the well of the gel.
           Note the samples in each well

   Loading a gel.

4. Run gel for 30 minutes for best clarity.
           Can use 15 minutes to see the results.
5. Observe gel when done, use a UV light, be careful to follow the manufactures recommendation when observing the gels.  Take pictures to keep a record.

   One of our many gels.

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Gene Cleaning

Purpose: Gene Cleaning removes contaminates produced during PCR.

Materials: QIA quick system (PB Buffer, PE Buffer, EB Buffer, filtration tube), product, microcentrifuge tube

Procedure:

1. Place the spin filtration column inside the collection tube.

   Insert the spin column inside the collection tube

2. Add 100 µL PB Buffer to the PCR tube, mix, transfer to spin column, spin for 1 minute at full speed.
3. Dump flow-through (collection tube), put filtration column back inside collection tube.
4. Add 750 µL PE Buffer to wash, spin for 1 minute at full speed.
5. Repeat step #3, spin dry for 1 minute at full speed.
6. Throw away collection tube, cut off top of filtration tube (secret step), place the filtration tube into a 1.5 mL microcentrifuge tube.
7. Add 40 µL EB buffer, make sure to add it to the very center of the filtration paper at the bottom of the filtration tube.  Incubate at Room Temperature for 1 minute.  Spin for 1 minute.
8. Throw away filtration column, run gel.

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Sequencing Reaction

Purpose: The sequence reaction includes a labeled nucleic acid that allows the sequence to be read as it flows through the sequencer.

Materials: 100% 2-Propanol (Isopropanol), PCR Samples, microcentrifuge tube, loading dye, DNA sequencing gel

Procedure:

1. Add 25 µL of 2-Propanol to the reaction tube, mix and then transfer to a labeled tube.
2. Incubate 10 minutes at Room temperature.  Centrifuge at full speed for 20 minutes.
3. Dump liquid by turning upside and touching a paper towel to get most of the liquid.
4. Resuspend pellet in 25 µL of 2-Propanol, incubate 20 minutes at room temperature and centrifuge at full speed for 15 minutes.
5. Dump liquid by turning upside and touching a paper towel to get most of the liquid.
6. Heat at 90°C until dry.  Minimum 5 minute.
7. Add 2.5 µL sequencing loading dye.  Put on ice for 3 minutes.
8. Centrifuge up to speed (max) for about 10 seconds.  Put back on ice until loaded.
9. Load the sample onto the sequencing gel.
10. Analyze the results.

    	v
    Standing in line for the sequencer.
    My turn to PLAY with the sequencer.

     

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Assessing the Sequence (BLAST)

Purpose: Here you will compare your results from the sequencer with known sequences of DNA.

Materials: Chromas 2 program (http://www.technelysium.com.au/chromas.html), sequences

Procedure:

1. Download the Chromas 2 software (there are other programs available).  We used the freeware, that is good for 60 days.
2. Open the program and load your sequence. 
3. Determine the location of your primer.  This is done by determining where the large peaks end.  Set the curser on the base and then go to the edit menu, then click on Set Left Cutoff (can use Alt L).  All of the bases to the left will be shaded in light yellow and will be ignored for determining DNA comparisons.
4. Now go through the sequence and replace any letter N's with a base, you can determine that by looking at the chromograms.  We prefer to use lower case letters to show that they are bases that we determined base on the chromogram.  You can change the amplification by moving the bar on the left up and down.  Save this sequence as your edited version.
5. Now go to file menu and run a Blast Search.  This will run a comparison to known genetic sequences a variety of "gene banks."  Now review the data.
6. For the protein sequence, go to the options menu, slide down to Show Translation, then chose All-Three Lines.  This shows all three protein sequences as determine where you start the codon reading.  Now go through the sequences, a * means a stop codon was seen and while amino acids are shown after this sequence, in reality, the protein will not be made beyond this point.
7. Choose the best sequence and ran a Blast Search as in step #5.  This will run a comparison to known protein sequences in a variety of "protein banks."  Now review the data.

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