GMO PCR Field Trip

Setup

Note: Set up 4 students per bench.

Equipment needed for each lab bench where students will work:

1. Goggles
2. Lab coats
3. Gloves
4. Waste beaker
5. Kimwipes
6. Ice bucket with crushed ice
7. p20 Pipetman (one per student)
8. p200 Pipetman (one per 2 students)
9. p1000 Pipetman (one per 2 students)
10. 2 boxes yellow tips
11. 2 boxes blue tips
12. Test tube racks and Sharpie pens

I. Genomic DNA Isolation

Reagents needed:

1. Wizard^® Magnetic DNA Purification System for Food (Promega Cat# FF3750)
2. 70% ethanol wash solution
3. Isopropanol (2-propanol)
4. Nuclease-free water
5. Assorted food products. BTCI uses a variety of processed (Cheetos and Doritos)
   and less-processed (cornmeal corn, or soybeans) foods, ground to coarse powder in a coffee grinder.

Equipment needed:

1. Tabletop centrifuge (capable of 13,000 x g)
2. Vortexer
3. 2.0ml microcentrifuge tubes
4. MagneSphere^® Technology Magnetic Separation Stand
   (Promega Cat# Z5332)
5. 65°C heat block or water bath

II. Polymerase Chain Reaction Amplification of CaMV35S Promoter Region

Reagents needed:

1. Nuclease-free water
2. Mineral oil in a dropper-bottle
3. 5 thin-walled PCR tubes
4. One 0.5ml microcentrifuge tube for preparing PCR master mix
5. The following reagents in the ice bucket:

Stocks	Quantity	Reagent	Aliquot Volume
Integrated DNA Technologies product diluted to 100pmol/μl	1	F Forward Primer (100 pmol/μl)	10 ul
Integrated DNA Technologies product diluted to 100pmol/μl	1	R Reverse Primer (100 pmol/μl )	10 ul
Promega product (M890A) from tube as-is	1	5x Taq PCR buffer	70 ul
Promega product (M829B) from tube as-is	1	Taq Pol (5 u/μl )	2 ul
Promega product (U151A) from tube as-is	1	dNTP (10mM total, 2.5 mM each nucleotide)	8 ul
Promega product (A351H) from tube as-is	1	MgCl2 (25mM)	40 ul
Isolated and Quantified Food DNA*	#	DNA Isolated from Food*	12 ul

* DNA templates:DNA templates can be either student or teacher-generated. For DNA isolated from less-processed foods, 25~50ng template should be sufficient for amplification. For highly-processed foods, DNA is usually degraded and the quantity required for amplification may be from two to tenfold higher, depending upon DNA quality.

Equipment Needed:

• Thermalcycler

III. Agarose Gel Electrophoresis of PCR Products

Reagents Needed:

1. 1x NaBorate running buffer (~0.5 liter per gel)
2. 250ml flasks with 2% agarose
   (2g agarose in 100ml makes enough for 2 gels; plan for ~40ml agarose/gel)
3. FOTO/Vision^TM DNA stain (12μl per gel)
4. Prepared 100bp DNA marker (8μl per gel)
   (To prepare marker, dispense 6μl marker from stock tube into a labelled, clear .5ml tube. Add 2μl FOTO/VisionTM DNA stain to the marker. One tube of marker is sufficient for one group)