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Published 20 October 2013

GMO PCR - Procedure

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Protocol for DNA extraction and PCR

I. Extraction of DNA

1. Preparation
1.1 Label 2 screw cap tubes with:
- non-GMO (-GMO)
- food sample (T)
and your group number!
1.2 Pipet 250 µl of homogenised InstaGene-Matrix from your tube labeled “IG” in both tubes!
There have to be the same amount of beads in both tubes!

2. Extraction of DNA from „non-GMO food“
2.1 Weigh out 1 g of certified non-GMO food and put it in a mortar!
2.2 Add 5 ml of distilled H2O!
2.3 Homogenise the mixture for about five minutes!
2.4 Again add 5 ml of H2Odist. and pestle until smooth enough to pipet the mixture!
2.5 Pipet 25 µl of the food mixture into the liquid of the screw cap tube labeled “-GMO”!
2.6 Recap tube and shake well by flicking the tube!

3. Extraction of DNA from food sample
3.1 Weigh out 1 g of food sample to be tested (T) and place it in the mortar!
3.2 Add 5 ml of H2Odist.!
3.3 Homogenise the mixture for about five minutes!
3.4 Again add 5 ml of H2Odist. and pestle until smooth enough to pipet the mixture!
3.5 Pipet 25 µl of the food mixture into the liquid of the screw cap tube labeled “T”!
3.6 Recap tube and shake well by flicking the tube!

4. Denaturation of enzymes at 95°C
4.1 Place both screw cap tubes in water bath of 95oC for 5 minutes!
4.2 Centrifuge the tubes for 5 minutes at 13000 rpm!
4.3 Put 50 µl of supernatant into two new screw cap tubes which have been labeled before!
Store tubes in refrigerator (8o C) until carrying out PCR reactions.

II. Preparation: PCR and electrophoresis in group work
According to their assignment, each group prepares a part of the PCR and the electrophoresis for all the other groups.

III. Preparation and carrying out of PCR

1. Preparation of PCR-tubes
1.1 Number your PCR-tubes 1-6!
1.2 Pipet the PCR-preparations according to the following table!
1.3 Mix your PCR-preparations by pipeting up and down!

1.4 Cool your PCR-preparations on ice until starting PCR!

2. Carrying out of PCR
Carry out the PCR according to the temperature program of the thermocycler!
Pay attention on the position of your samples in the thermocycler to avoid confusion! Make sure that your tubes are correctly closed!

IV. Evidence of PCR-products by electrophoresis (Agarose/PAGE)

1. According to your assignment give evidence of the PCR-products by carrying out Agarose-or Polyacrylamite-gel- electrophoresis!
2. According to your assignment dye your gels and analyse them!

Detection of PCR Products by Agarose Gel Electrophoresis

1. Preparation of the Gel Chamber
1.1. 1 g agarose is added to 33 ml TAE-Puffer in a 100 ml Erlenmeyer flask!
1.2. Boil the mixture in a microwave, shake it and boil it again shortly!
1.3. Let the agarose solution cool down till 55 °C (back hand test) and pour the gel!
1.4. After the gel has become solid, remove the metal rods of the electrophoresis chamber!
1.5. Fill the electrophoresis chamber with 270 ml TAE-Buffer to cover the gel!
1.6. Pull the comb!

2. Preparation of the Samples for the Gel Electrophoresis
2.1. Take your 6 PCR tubes out of the thermocycler!
2.2. Put your PCR tubes into your six tube adapters!
2.3. Pipet 10 µl Orange G loading dye (LD) in each of your 6 PCR tubes und mix them by gently snipping the tubes with your finger!

3. Filling the Gel Wells
3.1. Fill the gel wells from left to right with:
- 20 µl molecular weight ruler (MWR)
- 20 µl –GMO control with PMM (1)
- 20 µl –GMO control with GMM (2)
- 20 µl food sample (T) with PMM (3)
- 20 µl food sample (T) with GMM (4)
- 20 µl +GMO-DNA with PMM (5)
- 20 µl +GMO-DNA with GMM (6)

4. Running the Electrophoresis
4.1. Close the electrophoresis chamber and start the electrophoresis at 120 V!
4.2. Stop the electrophoresis when the stain Orange G has migrated to 1 cm before the end of the gel!

5. Making DNA Fragments Visible
5.1. Open the electrophoresis chamber and take out the sledge with the gel! Be careful the gel is very slippery! (Collect the TAE buffer, it could be used again!)
5.2. Transfer the gel in a staining tray and add 70 ml of staining solution (50x) for two minutes with gentle shaking!
5.3. Remove the staining solution after two minutes and destain it in warm tap water (40 - 50 °C) for 20 minutes with gentle shaking! Save the used stain for future use!

6. Evaluation
6.1. Identify the DNA bands in the gel with the help of the molecular weight ruler with the following lengths: 100 bp, 200 bp, 500 bp, 700 bp, 1000 bp!
6.2. Is your tested food sample (T) genetically modified? Prepare a short talk about your results!


There are files of the practical course GMO PCR.

Protocol for DNA extraction and PCR in pdf and word document and a ppt presentation of this experiment

PDF - 63.5 kb
Word - 5.5 Mb
PowerPoint - 1.4 Mb

Detection of PCR Products by Agarose Gel Electrophoresis in pdf and word document and a ppt presentation of this experiment

PDF - 57.6 kb
Word - 4.9 Mb
PowerPoint - 1.6 Mb