Protocol: Detection of Genetically Modified Organisms (GMO) – Vegetables
Pls. Follow the ordered steps.
1. Obtain a tube containing mashed GMO and Extraction Buffer from 56 C waterbath
(Before class instructor weighed out about 100 mg of GMO food and added 400 ul of
extraction buffer to break open cells)
2. Add 300 ul of NaCl solution
3. Mix well for 30 seconds by vortexing or vigorous tapping with fingers (finger-flick
technique)
4. Centrifuge at full speed for 15-30 mins (we’ll do this as a full class-all samples
together)
5. Remove the supernatant and transfer it to a fresh 1.5 mL microfuge tube.
6. Add an equal volume (approx. 700 ul) of isopropanol to your sample to precipitate the
DNA.
7. Incubate in the freezer for 40 mins-1 hr.
8. Centrifuge at full speed for 20 mins
9. Completely remove the supernatant
10. Add 1.5 mL 70% ethanol to wash the DNA. Slowly add the ethanol and then remove
it. Try not to disturb the pellet. If you disturb the pellet, then centrifuge 2 mins.
11. Discard the ethanol and dry pellet completely by placing the microfuge tube on a
paper towel face down
12. Dissolve the DNA pellet in 300 ul of 1X TE and keep sample on ice!
PCR Detection of GMO TAG
1. Tap the PCR tube to ensure the PCR reaction pellet is at the bottom of the tube. The
pellet contains nucleotides, buffer, magnesium and Taq enzyme.
2. Label the PCR tubes for the appropriate sample
3. To prepare a PCR reaction mix, add the following to the PCR pellet
DNA template from foodstuffs 5 ul
Primer mix (two primers)
20 ul
4. Gently mix the reaction tube. Before finishing be sure the PCR pellet is fully
dissolved
5. Place in PCR machine and cycle the reaction.
Initial denaturation 94 C 1 min
denaturation
annealing
extension
94 C 1min
63 C 1 min
72 C 1 min
For 50 cycles
Final extention 72 C 1 min