practical training: proof of a gentechnical variation of foods with pcr
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Practical training: Proof of a gentechnical variation of foods with PCR. 60``, 94 ° Denaturation. 60``, 55°C Annealing. 120``, 94°C Denaturation. 40 cycles. 120``, 72 °C Elongation. 10`, 72 °C Completing. Principle preperation of a PCR. I. Extraction of DNA. 1. Preparation. - PowerPoint PPT PresentationTRANSCRIPT
Practical training:Proof of a gentechnical variation of
foods with PCR
Principle preperation of a PCR
10`, 72 °CCompleting
60``, 94 ° Denaturation
40 cycles
120``, 94°C Denaturation
60``, 55°CAnnealing
120``, 72 °CElongation
I. Extraction of DNA
1. Preparation
1.1 Label 2 screw cap tubes on the top with:
- non-GMO (- GMO)- food sample (T)and your group number!
1.2 Pipet 500 µl of homo-genised InstaGene-Matrix into each tube!
There have to be the same amount of beads in both tubes!
supernatant
beads
2. Extraction of DNA from „non-GMO food“
2.1 Weigh out 1 g of certified non-GMO food and put it into the
mortar!
2.2 Add 5 ml of distilled H2O!
2.3 Homogenise the mixture for about five minutes!
2.4 Add 5 ml of H2Odist. and pestle
until smooth enough to pipet the mixture!
2. Extraction of DNA from „non-GMO food“
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!
To avoid contamination with „+GMO DNA“ „-GMO material“ should be extracted first!
Mortar and pestle should be cleaned with a detergent and rinsed out with distilled H2O!
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.!
Please use gloves!!!
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. Extraction of DNA from food sample
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 newtubes which have been labeled before!
Pay attention: Only take the supernatant without the InstaGene beads by removing of the food control DNA and the test food DNA!
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!
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!
2. Carrying out of PCR
Cycler-Program
Overview of the tubes in the cycler
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!
Filling thegelslots