I. Extraction of DNA
1. Preparation
1.1
1.2
Label 2 screw cap tubes with:
- non-GMO (-GMO)
- food sample (T)
and your group number!
Pipet 250 µl of homogenised
InstaGene-Matrix from your tube labeled
“IG” in both tubes!
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.
There have to be the same
amount of beads in both
tubes!
2. Extraction of DNA from „non-GMO
food“
2.1
2.2
2.3
2.4
2.5
2.6
Weigh out 1 g of certified non-GMO food
and put it in a mortar!
Add 5 ml of distilled H2O!
Homogenise the mixture for about five
minutes!
Again add 5 ml of H2Odist. and pestle until
smooth enough to pipet the mixture!
Pipet 25 µl of the food mixture into the
liquid of the screw cap tube labeled
“-GMO”!
Recap tube and shake well by flicking the
tube!
3. Extraction of DNA from food sample
3.1
3.2
3.3
3.4
3.5
3.6
Weigh out 1 g of food sample to be tested
(T) and place it in the mortar!
Add 5 ml of H2Odist.!
Homogenise the mixture for about five
minutes!
Again add 5 ml of H2Odist. and pestle until
smooth enough to pipet the mixture!
Pipet 25 µl of the food mixture into the
liquid of the screw cap tube labeled “T”!
Recap tube and shake well by flicking the
tube!
4. Denaturation of enzymes at 95°C
4.1
4.2
4.3
Place both screw cap tubes in water bath of
95oC for 5 minutes!
Centrifuge the tubes for 5 minutes
at 13000 rpm!
Put 50 µl of supernatant into two
new screw cap tubes which have been
labeled before!
III. Preparation and carrying out of
PCR
1. Preparation of PCR-tubes
1.1
1.2
1.3
Number your PCR-tubes 1-6!
Pipet the PCR-preparations according to the
following table!
Mix your PCR-preparations
by pipeting up and down!
Nr. Master Mix
DNA
1
2
3
4
5
6
10 µl –GMO(control)
10 µl –GMO (control)
10 µl food sample (T)
10 µl food sample(T)
10 µl +GMO (control)
10 µl +GMO (control)
10 µl PMM
10 µl GMM
10 µl PMM
10 µl GMM
10 µl PMM
10 µl GMM
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.
2.
According to your assignment give evidence
of the PCR-products by carrying out
Agarose-or Polyacrylamite-gelelectrophoresis!
According to your assignment dye your gels
and analyse them!