Protocol

The Bio-Rad GMO Investigator Kit was used in this experiment.

Try 1 – Preparing the Negative Control:

One oat was placed in a mortar and ground with a pestle. Sterilized water was added until the mixture was smooth enough to pipet. 50μL of the oat mixture was pipetted into a 2mL tube labeled “Non-GMO”. The tube was spun in a microfuge for 2 minutes. The supernatant was discarded and the cell pellet was re-suspended. The re-suspended oat pellet was pipetted into a new microfuge tube that contained 300μL of 10% Chelex. That tube was vortexed for one minute to mix the contents, and then was placed in a 95-degree Celsius heating bath for 10 minutes. The tube was removed from the hot water bath and spun in a microfuge for 2 minutes. 50μL of the supernatant containing the oat DNA was pipetted into a sterile microfuge tube labeled “negative control”.

making the neg control

Trial 1 – PCR Reactions:  

Four PCR tubes were labeled 1-4 and filled with the contents listed below:

tubes 1

The 4 tubes were placed in a PCR machine that followed this process:

  • 94 degrees for 2 mins
  • 40 cycles of:
  • 94 degrees for 1 min
  • 59 degrees for 1 min
  • 72 degrees for 2 min
  • 72 degrees for 10 mins
  • Hold at 15 degrees

After PCR was completed, the 6 tubes were held at 4 degrees Celsius in a freezer.

Trial 1 – Running the Gel:
A 0.7% agarose gel was made. 4 μL of 6x loading dye was added to 20μL of each PCR reaction. In lane 1, 10μL of 10 log ladder was loaded. In lanes 2-5, PCR reactions 1-4 with loading dye were loaded. The gel was run on high for one hour. Then, the gel was stained with Sybr Gold and analyzed under a UV light.

annotated-gel-trial-1-e1491243497115.jpg

**the gel yielded inconclusive results, so the PCR reactions were tweaked and the process was repeated**

Trial 2 – PCR Reactions: 

Six PCR tubes were labeled 1-6 and filled with the contents listed below:

Try 2 PCR contents

Two master mixes were made: one for the positive control and one for the negative control. The Positive MM contained 60μL sterilized H2O, 10μL 10x buffer, 2μL DNTPs, and 0.5μL taq. The Negative MM contained 72μL sterilized H2O, 10μL 10x buffer, 2μL DNTPs, and 0.5μL taq.

The 6 tubes were placed in a PCR machine that followed this process:

  • 94 degrees for 2 mins
  • 40 cycles of:
  • 94 degrees for 1 min
  • 59 degrees for 1 min
  • 72 degrees for 2 min
  • 72 degrees for 10 mins
  • Hold at 15 degrees

After PCR was completed, the 6 tubes were held at 4 degrees Celsius in a freezer.

Trial 2 – Running the Gel:

Another 0.7% agarose gel was made. 5 μL of 6x loading dye was added to each PCR reaction (tubes 1-6). In lane 1, 10μL of 2 log ladder was loaded. Lane 2 was left empty. In lanes 3-8, PCR reactions 1-6 were loaded in that order. The gel was run on high for 45 minutes. Then, the gel was stained with Sybr Gold and analyzed under a UV light.

trial 2 loading

Try 2 – Preparing the Negative Control:

A couple oats were placed into a 2mL tube. Using a mini pestle and vortex, the oats were grinded into fine pieces. Then, 500μL of 1% saline solution was added to the tube. The tube was centrifuged in a microfuge for 2 minutes. To grind the oat mixture more, a mini pestle and vortex were used again. The tube was centrifuged in a microfuge for another 2 minutes. The oat mixture was vortexed to mix the oat fiber, DNA, and saline. 100μL of the oat mixture was pipetted into 300μL of chelex in a new 2mL tube. The new tube containing the oat mixture and chelex was vortexed for 1 minute. The tube was then placed in a 95 degree Celsius heating bath for 10 minutes. Then the tube was spun in a microfuge for 2 minutes. Finally, 50μL of the supernatant containing the DNA was pipetted into a sterile 2mL tube.

try 2 dna extraction from oats

**the same protocol of “Trial 2 – PCR Reactions” and “Trial 2 – Running the Gel” were followed**

DNA Extraction of Test Foods: 

**the same protocol of “Try 2 – Preparing the Negative Control” was followed, except each cereal was substituted for the oat negative control**

PCR Reactions of Test Foods:  

Six PCR tubes were labeled and filled with the contents listed below:

test food PCR contentsThe MM contained 60μL sterilized H2O, 10μL 10x buffer, 2μL DNTPs, and 0.5μL taq.

The 6 tubes were placed in a PCR machine that followed this process:

  • 94 degrees for 2 mins
  • 40 cycles of:
  • 94 degrees for 1 min
  • 59 degrees for 1 min
  • 72 degrees for 2 min
  • 72 degrees for 10 mins
  • Hold at 15 degrees

After PCR was completed, the 6 tubes were held at 4 degrees Celsius in a freezer.

Loading the Gel with Test Foods: 

A 0.7% agarose gel was made. 4 μL of 6x loading dye was added to 20μL of each PCR reaction. In lane 1, 10μL of 10 log ladder was loaded. Lane 2 was left empty. In lanes 3-8, PCR reactions of the positive control and test foods A, B, C, D and E with loading dye were loaded. The gel was run on high for one hour. Then, the gel was stained with Sybr Gold and analyzed under a UV light.

test food gel order.JPG

Dilution of Rubisco Primers Calculations: 

primer calculations

PCR Reactions of Test Foods with Rubisco and Dr. Todd’s GMO Primers:  

Ten PCR tubes were labeled and filled with the contents listed below:

New Primers PCR reaction contents

The MM contained 180μL sterilized H2O, 30μL 10x buffer, 6μL DNTPs, and 1.5μL taq.

The 10 tubes were placed in a PCR machine that followed this process:

  • 94 degrees for 2 mins
  • 40 cycles of:
  • 94 degrees for 1 min
  • 59 degrees for 1 min
  • 72 degrees for 2 min
  • 72 degrees for 10 mins
  • Hold at 15 degrees

After PCR was completed, the 10 tubes were held at 4 degrees Celsius in a freezer.

Loading the Gel with PCR Reactions of Test Foods with Rubisco and Dr. Todd’s GMO Primers:  

A 0.7% agarose gel was made. 4 μL of 6x loading dye was added to 20μL of each PCR reaction. In lane 1, 10μL of 10 log ladder was loaded. Lane 2 was left empty. In lanes 3-7, PCR reactions 1-5 were loaded. Lane 8 was left empty. In lanes 9-13, PCR reactions 6-10 were loaded. The gel was run on high for one hour. Then, the gel was stained with Sybr Gold and analyzed under a UV light.

gel order for rubiscogel order for rubisco 2

Gradient PCR Reactions of Cereal D with Rubisco Primers:

The master mix below was prepared:

1x (uL) 10x (uL)
H2O 19 190
10x 2.5 25
DNTP .5 5
Forward Primer (Rubisco) 1 10
Reverse Primer (Rubisco) 1 10
Taq .125 1.25
*take out one reaction for neg control*
DNA (Cereal D) 1 9
Total 25  250
A 43-60 degree Celsius gradient was ran out on PCR. The eight reactions were placed, staring in column 3 and ending in column 10, in the PCR machine. The samples were annealed from 44.5-58.6 degree Celsius.
gradient location

Location of Samples in PCR

The PCR gradient was ran following this procedure:
gradient pcr reaction protocol

Gradient PCR Reactions of Cereal D with GMO Primers:

The master mix below was prepared:

1x (uL) 10x (uL)
H2O 19 190
10x 2.5 25
DNTP .5 5
Forward Primer (Rubisco) 1 10
Reverse Primer (Rubisco) 1 10
Taq .125 1.25
*take out one reaction for neg control*
DNA (Cereal D) 1 9
Total 25  250

A 46-60 degrees Celsius gradient was ran out on PCR. The eight reactions were placed, staring in column 4 and ending in column 11, in the PCR machine. The samples were annealed from 48.3-59.7 degrees Celsius.

gradient of gmo

Location of Samples in PCR

The PCR gradient was ran following this procedure:
gmo gradient pcr procedure

Optimal Temperature PCR Reactions of Test Foods with Rubisco and Dr. Todd’s GMO Primers:  

A master mix for 14 reactions was made and included 210μL (new) water, 35μL 10x buffer, 7μL dNTPs, and 1.75μL taq. The master mix was separated into two equal parts and add 7μL of the Rubsico primers into one tube of master mix and add 7μL of the GMO primers into the second tube of master mix. 1μL of each test food was pipetted into separate PCR tubes twice; therefore, there were two tubes containing 1μL of each test food.

The following reactions were made:

optimal temp pcr contents.JPG

The 12 tubes were placed in a PCR machine that followed this process:

  • 95 degrees for 2 mins
  • 35 cycles of:
    • 95 degrees for 1 min
    •  58 degrees for 1 min
    • 68 degrees for 2 min
  • 68 degrees for 10 mins
  • Hold at 4 degrees

DNA Extraction of Cereals A and B:

One flake of a cereal type was placed into a 2mL tube. Using a mini pestle and vortex, the flake was grinded into fine pieces. Then, 500μL of 1% saline solution was added to the tube. The tube was centrifuged in a microfuge for 2 minutes. To grind the flake mixture more, a mini pestle and vortex were used again. The tube was centrifuged in a microfuge for another 2 minutes. The flake mixture was vortexed to mix the oat fiber, DNA, and saline. 100μL of the oat mixture was pipetted into 300μL of chelex in a new 2mL tube. The new tube containing the flake mixture and chelex was vortexed for 1 minute. The tube was then placed in a 95 degree Celsius heating bath for 10 minutes. Then the tube was spun in a microfuge for 2 minutes. Finally, 50μL of the supernatant containing the DNA was pipetted into a sterile 2mL tube.

try 2 dna extraction from oats

A master mix for 10 reactions was made and included 150μL (new) water, 25μL 10x buffer, 5μL dNTPs, and 1.25μL taq. The master mix was separated into two equal parts and add 5μL of the Rubsico primers into one tube of master mix and add 5μL of the GMO primers into the second tube of master mix. 1μL of each test food was pipetted into separate PCR tubes twice; therefore, there were two tubes containing 1μL of each test food.

pcr reactions CDE

The 8 tubes were placed in a PCR machine that followed this process:

  • 95 degrees for 2 mins
  • 35 cycles of:
    • 95 degrees for 1 min
    •  58 degrees for 1 min
    • 68 degrees for 2 min
  • 68 degrees for 10 mins
  • Hold at 4 degrees

Making the PAGE Gel:

1.5uL Accugel was pipetted into a sterile tube. Then, 100μL of 50x TAE was added. 3.5mL of sterile water was put into into the tube. A 10% APS solution was made by dissolving 0.3g of APS in 3mL water. Then, 50μL of 10% APS sol into the original tube. 5μL of TMED was added to the tube.

New DNA Extraction Procedure:

This procedure was followed for all 5 cereals (A, B, C, D and E). First, a quarter of each flake was placed into separate sterile tubes. Each tube was vortex and the cereals were crushed using a mini pestle until it was a fine powder. 300μL of a 1% saline solution was added to each tube containing the ground cereal. Each tube was vortexed and then spun in a microfuge for 2 minutes. All of the supernantent for each cereal was pipetted into new tubes, each containing 300μL chelex. The new tubes were then vortexed for two minutes and then put in a 95 degrees in hot water bath for 10 mintues. After the water bath, the tubes were spun for two minutes and 50μL of each supernatant was pipetted into new tubes. All of the tubes were then placed into the freezer.

new dna extraction protocol

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