1 / 17

Practical training: Proof of a gentechnical variation of foods with PCR

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.

lucas-oliver
Download Presentation

Practical training: Proof of a gentechnical variation of foods with PCR

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Practical training:Proof of a gentechnical variation of foods with PCR

  2. 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

  3. I. Extraction of DNA

  4. 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

  5. 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!

  6. 2. Extraction of DNA from „non-GMO food“ 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!

  7. 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!

  8. 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!!!

  9. 3. Extraction of DNA from food sample 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!

  10. 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!

  11. 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 tubes which have been labeled before!

  12. Pay attention: Only take the supernatant without the InstaGene beads by removing of the food control DNA and the test food DNA!

  13. 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.

  14. III. Preparation and carrying out of PCR

  15. 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!

  16. Cycler-Program 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 surethat your tubes are correctly closed! Overview of the tubes in the cycler

  17. IV. Evidence of PCR-products by electrophoresis (Agarose/PAGE) • According to your assignment give evidence of the PCR products by carrying out Agarose- or Polyacrylamite-gel- electrophoresis! • According to your assignment dye your gels and analyse them! Filling the gelslots

More Related