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K-12
Programs
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Isolation of genomic DNA from food material Purpose:
Protocol: Genomic DNA Isolation from foodMaterials to Be Supplied: Disposable gloves Pipettes Pipette Tips (Promega) Table top centrifuge (capable of 13,000 x g.) Vortexer Wizard® Magnetic DNA Purification System for Food (Promega Cat. # FF3750) 2ml microcentrifuge tubes 95% or 100% Ethanol 70% Ethanol Wash Solution Isopropanol (2-Propanol) Nuclease-Free Water (Promega Cat. # P1193) MagneSphere® Technology Magnetic Separation Stand (Promega Cat. # Z5331 or Z5332) 65oC heat block or water bath Ice in a bucket
1. Weigh out 200mg food material and transfer to a 2ml microcentrifuge tube. · Crush food materials such as DoritosÒ Tortilla Chips before measuring. 2. Tilt the tube to the side so that the food material is covering the side of the tube. Add 500ul Lysis Buffer A to the food material. Cap the tube and vortex vigorously.\ 3. Add 5ul RNase A to the tube. Cap the tube and vortex vigorously. 4. Add 250ul Lysis Buffer B to tube. Cap the tube and vortex vigorously for 10-15 seconds. Place the tube on it’s side. 5. Incubate the tube at room temperature for 10 minutes. (22-25oC). 6. Add 750ul blue Precipitation Solution. Cap the tube and vortex vigorously. · For most samples, the mixture will be a light green color at this point. The sample should be evenly suspended. If not, vortex or mix with a pipette tip. 7. Spin for 10 minutes in a microcentrifuge at maximum speed. (13,000 x g) 8. Transfer the supernatant to a fresh 2ml microcentrifuge tube. There will be some non-digested food material in the bottom of the tube. Dispose the tube once the lysate has been removed to a new tube. · For most samples (corn meal and DoritosÒ Tortilla Chips) the supernatant will be the green colored clear phase. For the Lecithin, the supernatant is the milky mint green phase. If there is floating material on top of the liquid phase, carefully pipette under it. 9. Mix the bottle of MagneSil® Paramagnetic Particles (PMPs) by shaking for 15-30 seconds to make sure that the brown PMPs are thoroughly resuspended. Add 50ul of resuspended particles to the lysate now in the clean 2ml microcentrifuge tube. 10. Add 1ml Isopropanol to the tube containing the lysate and PMPs. Cap the tube and invert the tube in your hand 10-15 times to mix. 11. Incubate the tube at room temperature for 5 minutes, mixing the tube by inversion by hand occasionally. 12. Insert the tube into the magnetic separation stand and leave in place for 1 minute. · You will see the PMPs move to the side of the tube closest to the magnetic stand. 13. Leave the tube in the magnetic stand. Once all the PMPs have collected on the side of the tube, remove the cap and remove the liquid phase by pipette and discard. 14. Remove the tube from the stand. Add 250ul Lysis Buffer B to the particles. Cap the tube and mix by inversion by hand. 15. Place the tube back into the magnetic stand. 16. Leave the tube in the magnetic stand. Once all the PMPs have collected on the side of the tube, remove the cap and remove the liquid phase by pipette and discard. 17. Remove the tube from the stand. Add 1ml 70% ethanol wash solution to the particles. Cap the tube and mix by inversion by hand. 18. Place the tube back into the magnetic stand. 19. Repeat steps 16-18 two more times. 20. Leave the tube in the magnetic stand. Once all the PMPs have collected on the side of the tube, remove the cap and remove the liquid phase by pipetting and discard. 21. Leave the tube in the magnetic stand. Leave the cap off the tube. Let the tube sit at room temperature for 5 minutes to allow the alcohol to evaporate. 22. Remove the tube from the stand. Add 100ul of Nuclease-free water. Incubate at 65oC for 5 minutes. 23. Place the tube back into the magnetic stand. 24. Leave the tube in the magnetic stand. Once all the PMPs have collected on the side of the tube, remove the cap and remove the liquid to a new 2ml microcentrifuge tube. Discard the tube containing the PMPs. This final 100ul in the new microcentrifuge tube contains your genomic DNA isolated from the food material. Store your isolated DNA on ice. Genomic DNA can be stored for up to 2 months at 4 oC (refrigerator), although some degradation may take place. For longterm storage use a freezer. PCR Detection of Genetically Modified Foods (GMO) Field Trip Protocol
Purpose:
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 (10 mM) 35S Reverse Primer (10 mM) dNTPs MgCl2 PCR buffer Thermal cycler Ice in a bucket Agarose Sodium Borate Buffer Electrophoresis Power Supply Electrophoresis Chamber Gel Casting Tray 12 Well Gel Comb 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 CC- Corn Chips M- Corn Meal (+) - Certified GMO Corn (- ) – Negative Control 2. Assemble a PCR master mix of the following required PCR reagents in a 1.5 ml tube. Table 1. PCR Reaction
3. Pipet 48 mL master mix into the six labeled 0.5ml Thin Walled PCR tubes. 4. Add 2 mL template/sample food DNA to each corresponding PCR tube. 5. Place tubes in Thermal Cycler and begin cycling using the following conditions. PCR Amplification of Food DNA Twenty seven 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: Temperature PCR Step Time Number of Cycles Purpose 94°C Denaturation 3 minutes 1 Ensures that all 54° C Primer Annealing 40 seconds the template DNA 72° C Extension 60 seconds molecules are denatured. 94° C Denaturation 20 seconds 25 Amplification of 55° C Primer Annealing 40 seconds target DNA sequence 72° C Extension 60 seconds (CaMV35S promoter) 94° C Denaturation 20 seconds 1 Gives DNA polymerase 54° C Primer Annealing 40 seconds extra time to complete 72° C Extension 3 minutes all of the PCR products.
A total of twenty-seven 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 molten gel has cooled to around 55º 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 you gel casting tray by raising the buffer dams and securing them in place.
7. Insert a 12 well comb into the upper groove on the casting tray.
8. Pour approximately 35 ml of molten agarose into the casting tray, and let cool.
9. Label 6 clean 0.5ml tubes, and add 2mL of 6X loading dye to each tube.
10. Pipet 10mL of each corresponding sample into your labeled tubes and mix by pipetting.
11. Add about 325-350 mls of 1X sodium borate buffer to the electrophoresis chamber, buffer should just cover the gel.
12. Load 10 ml 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.
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. Drain off as much buffer as possible. Remember that the gel contains EtBr. If your gloves are wet from the buffer or handling the gel, change them before touching anything else in the lab. Place the gel on the UV light box to visualize the DNA and to photograph it.
Gel Loading Diagram
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Contact: Barbara Bielec, 608-273-9737 |
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