DNA-Fingerprint1 Detection of PCR Products by Agarose Gel Electrophoresis.

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DNA-Fingerprint1 Detection of PCR Products by Agarose Gel Electrophoresis

DNA-Fingerprint2 1. Preparation of the Gel Chamber

DNA-Fingerprint3 1. Preparation of the Gel Chamber 1.1 1g 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! 1.3Let the agarose solution cool down till 55 °C (back hand test) and pour the gel!

DNA-Fingerprint4

5 1.4After the gel has become solid, remove the metal rods of the electrophoresis chamber! 1.5Fill the electrophoresis chamber with 270 ml TAE-Buffer to cover the gel! 1.6Pull the comb! 1. Preparation of the Gel Chamber

DNA-Fingerprint6 2. Preparation of the Samples for the Gel Electrophoresis

DNA-Fingerprint7 2. Preparation of the Samples for the Gel Electrophoresis 2.1Take your 6 PCR tubes out of the thermocycler! 2.2Put your PCR tubes into your six tube adapters!

DNA-Fingerprint8

9 2.3Pipet 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! 2. Preparation of the Samples for the Gel Electrophoresis

DNA-Fingerprint10 3. Filling the Gel Wells

DNA-Fingerprint11 3. Filling the Gel Wells 3.1Fill 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)

DNA-Fingerprint12 4. Running the Electrophoresis

DNA-Fingerprint13

DNA-Fingerprint14 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!

DNA-Fingerprint15 5. Making DNA Fragments Visible

DNA-Fingerprint16 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!

DNA-Fingerprint17 5.3Remove the staining solution after two minutes and destain it in warm tap water ( °C) for 20 minutes with gentle shaking! Save the used stain for future use! 5. Making DNA Fragments Visible

DNA-Fingerprint18 6. Evaluation

DNA-Fingerprint19 6. Evaluation 6.1Identify 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!

DNA-Fingerprint20 6.2Is your tested food sample (T) genetically modified? Prepare a short talk about your results! 6. Evaluation