PCR Detection of Genetically Modified Foods (GMO) Field Trip

Purpose

• Amplification of CaMV35S promoter DNA fragment from purified food samples

Protocol: GMO Foods PCR

Materials to Be Supplied

• Disposable gloves
• Pipettes
• Pipette Tips
• Table top centrifuge (capable of 13,000 x g.)
• 1.5 ml Tubes
• 0.5 ml Tubes
• Thin Walled PCR Tubes
• Nuclease-Free Water
• 35S Forward Primer (100 μM)
• 35S Reverse Primer (100 μM)
• dNTPs
• MgCl₂
• PCR buffer
• Thermal cycler
• Crushed ice in a bucket
• Agarose
• Sodium Borate Buffer
• Ethidium Bromide
• Electrophoresis Power Supply
• Electrophoresis Chamber
• 100bp DNA Ladder
• Gel Casting Tray
• Six-Well Gel Comb
• 6x Loading Dye

1. Label five 0.25 ml PCR tubes with your group number or your initials.  Label your tube with the name of your food sample.One person from each table of four will be responsible for one food product isolation, so label your PCR tubes as listed below.

   D - Doritos
   C - Cheetos
   CM - Corn Meal
   (+) - Soybean Positive Control
   (–) - Negative Control

2. Assemble a PCR master mix of the following required PCR reagents in a 1.5 ml tube.


Table 1. PCR Reaction

for each PCR Reaction	Components	6X Master Mix (you prepare this)
21.75μl	Nuclease-Free H2O	130.5μl
10.0μl	5X Taq PCR Buffer	60.0μl
5.0μl	MgCl2	30.0μl
1.0μl	dNTPs (2.5mM each)	6.0μl
1.0μl	Forward Primer (100pmol/μl)	6.0μl
1.0μl	Reverse Primer (100pmol/μl)	6.0μl
0.25μl	Taq Polymerase	1.5μl
use 40μl per reaction		240μl Total

3. Pipet 40μL master mix into the six labeled 0.5ml Thin Walled PCR tubes.
4. Add 10μL template/sample food DNA to each corresponding PCR tube, adding 10μl nuclease-free water to negative control tube in place of template DNA.
5. Place tubes in Thermal Cycler and begin cycling using the following conditions.

PCR Amplification of Food DNA

Twenty-nine cycles of PCR will be used to amplify the 35S promoter sequence.  Your instructor will program the thermal cycler to carry out the following temperature changes:

A total of twenty-nine PCR cycles will be completed by the thermal cycler and will take 2 hours.

Analyze results on a 2% agarose gel. ( The CAMV 35s Promoter is ~195 bp)

Gel Electrophoresis

1. Weigh out 2 g agarose, and put in a 250ml Erlenmeyer flask.
2. Measure 100ml of 1X Sodium Borate Buffer in a graduated cylinder
3. Pour the 100ml of 1X Sodium Borate Buffer in to the Erlenmeyer flask containing the agarose and gently swirl.  This will make enough agarose for 2+ gels.
4. Heat the agarose in the microwave until the solution is clear, and no more agarose is visible.
5. Once the agarose has cooled to 55°C, an instructor will add 5ul Ethidium Bromide
   (CAUTION: ETHIDIUM BROMIDE IS A MUTAGEN. HANDLE THE MOLTEN GEL CAREFULLY!!! AND WEAR PROPER PROTECTION AND CHANGE GLOVES AFTER POURING YOUR GEL.)
6. Assemble your gel casting tray by raising the buffer dams and securing them in place.  Insert a 6-well comb into the upper groove on the casting tray. 
7. Pour approximately 35 ml molten agarose into the casting tray, and let cool.
8. Label five clean 0.5ml tubes and add 2μl 6x Loading Dye to each tube.
9. Pipet 10μl of each corresponding sample into your labeled tubes and mix by pipetting.
10. Add about 325 to 350 ml 1x sodium borate running buffer to the electrophoresis chamber. The buffer should just cover the gel.
11. Load 10μl of each sample with dye into the appropriate wells of the gel - look at the diagram below. You will be provided pre-dyed 100bp DNA ladder for Lane 1.
12. Load 10μl of each sample with dye into the appropriate wells of the gel - look at the diagram below.  You will be provided with pre-dyed 100bp ladder for lanes 1 & 7.   Lane 8 is a non-amplified (no PCR) sample of food DNA.

Gel Loading Diagram

13. Place the lid on the gel box; connect the electrodes to the power supply. Make sure that the black wire goes into the black plug and the red into red.
14. Turn on the power supply and set it at 220 V. Bubbles at the electrodes indicate that the current is running. The gel will run for 20 minutes.
15. After the gel has run, remove it from the gel box. Remember that the gel contains ethidium bromide! If your gloves are wet from the buffer or handling the gel, change them before touching anything else in the lab. Drain off as much buffer as possible. Place the gel on the UV light box to visualize the DNA and to photograph it.