GMO LABORATORY PROCEDURE

DAY 1: ISOLATE DNA FROM SOY AND FOOD PRODUCTS

TODAY’s STARTING POINT
/ TODAY’s ENDING POINT

Please note: Each group of two will select EITHER wildtype or Roundup Ready soy OR a food product. Note the product you are working with______and be sure to label all tube with your assigned lab number______

The protocol below is written for each group to analyze two samples (soy and food).

1.
/ TUBE #1: SOY LEAVES
Cut two pieces of soy leaf 1/4 inch in diameter. Putboth pieces into a 1.5 ml tube. Label with soybeantype and your group number.
TUBE #2: FOOD
Crush a small amount of foodon a piece of paper. Put crushed food into a 1.5 mltube about halfway to the 0.1 ml mark. Label withthe food type and your group number.
2.
/ Add 100 l of Edward’s buffer to each tube.
3.
/ Forcefully grind the plant tissue or food product for 1 minute.
4.
/ Add 900 l of Edward'sbuffer to each tube.
5.
/ Flick the tubes for 5 seconds to mix things up a bit.
6.
/ Place boiling caps on samples. Boil the samples for 5 minutes in the heat block.
7.
/ Centrifuge the samples for 2 minutes to pellet cell and food debris.
8.
/ Put 350 l of each supernatant into new tubes.
Labe the new tubes carefully and throw out the old tubes so you don’t get confused.
9.
/ Add 400 l of isopropanol to each tube.Mix by turning the tubes upside-down several times. Once mixed, leave the tubes at room temperature for 3 minutes.
10.
/ Centrifuge for 5 minutes. Place tubes in the centrifuge with the cap hinges pointing up.
11.
/ Pour off the supernatant from each tube.
12.
/ Completely remove the remaining liquid with a pipette set at 100 l.
13.
/ Air dry the pellets for 10 minutes.
14.
/ Add 100 l of distilled water to each tube.
15.
/ Pipette the liquid in and out. Try to wash down the side of the tube underneath the hinge; this is where the DNA is. Let the tubes sit for 5 minutes.
16.
/ Centrifuge the tubes for 1 minute.
17.
/ Store your samples in the freezer. Make sure the tubes are still clearly labeled.

DAY 2: AMPLIFY DNA BY PCR

35S PROMOTER: Only found in genetically modified foods
Please note: Each group will set up one PCR reaction for 35S. Please label tube with your group number and 35S.
35S PCR TUBE #1: SOY DNA
Label PCR tube with your group number and 35SSOY DNA (RR or NRR)
Example: M2 5 35S NRR
35S PCR TUBE #2: FOOD DNA
Label PCR tube with your group number and 35SFOOD DNA (F).
Example: M2 5 35S F
/ Add 23l of the 35S primer/loading dye mix to each tube. Allow 3 minutes for the PCR bead to dissolve.
/ Add 3.0l of FOOD DNA (from Part I) to the PCR tube labeled “FOOD DNA”
Add 3.0l of SOY DNA (from Part I) to the PCR tube labeled “SOY DNA”
TUBULIN: Found in everything
Please note: Each group will set up one PCR reaction for Tubulin. Please label tube with your group number and T for Tubulin.
Tubulin PCR TUBE #1: SOY DNA
Label PCR tube with your group number and TubulinSOY DNA (RR or NRR)
Example: M2 5 Tub NRR
Tubulin PCR TUBE #2: FOOD DNA
Label PCR tube with your group number and TubulinFOOD DNA (F).
Example: M2 5 Tub F
/ Add 23.0l of the Tubulin primer/loading dye mix to each tube. Allow 3 minutes for the PCR bead to dissolve.
/ Add 3.0l of FOOD DNA (from Part I) to the PCR tube labeled “FOOD DNA”
Add 3.0l of SOY DNA (from Part I) to the PCR tube labeled “SOY DNA”
/ Bring all four PCR tubes to the thermocycler or store on ice (up to 24 hours) until ready.