Una delle attività svolte dai ragazzi del Liceo Scienze Applicate "M.Hack" presso il CREA di Turi nel laboratorio di Biologia Molecolare è stata quella dell'estrazione del DNA dalle giovani foglie di vite. I ragazzi hanno seguito e realizzato con passione e coinvolgimento le attività, come dimostra il report da loro redatto in lingua italiana e inglese. Un encomio ai ragazzi per l'eccellente lavoro svolto ed un ringraziamento particolare allo staff del CREA ed alla prof.ssa Cinzia Montrone per la supervisione dell'elaborato.

1. A.S. 2015/2016 CLASSI TERZE LICEO Responsabile: Prof.ssa Laura Persico
DNA EXTRACTION FROM VEGETAL MATRIX
Activity performed at Turi research unit (BA) of:

2. PREPARATION OF SAMPLES
 Sampling of grapevine leaves and their placing in numbe-
red aluminium foils.
 Weighing of the samples by an analitic scale and inserting
of a specific amount in Eppendorf test tubes together
with tungsten carbide beads.

3. The DNA extraction of a plant cell requires additional steps
compared to those of an animal one since the former has an
external membrane called cell wall, that improves the structu-
re and the nutrition of the plant.
Breaking of Cell Wall
 Freezing of samples by liquid nitrogen, to break the cell wall.
 Tissue mechanical breaking by a Tissue Lyser. With horizon-
tal oscillations, it shakes the beads in the tubes, allowing the
laceration of the leaves.

4. After the elimination of the cell wall, the procedure goes on
with the lysis of cell membrane:
 Addition of a AP1+RNase buffer mix to degrade and eliminate
RNA;
 Use of vortex to mix vigorously the content of the tubes;
 Incubation in water bath for 10 minutes with a temperature of
65°C, inverting the tubes every 2-3 minutes.
Cell lysis

5. Precipitation and separation of
proteins and polysaccharides
 Addition of Buffer AP2 (130µl) and mixing;
 Incubation in ice for 5 minutes;
 Arrangement in centrifuge for 5 minutes at the speed of
14000 rpm.

6. Removal of precipitates and
cell debris
 The solution is transferred in new tubes (lilac spin co-
lumn) provided with specific filters that hold the cell de-
bris allowing the precipitation of DNA;
 Arrangement in centrifuge for 2 minutes at the speed of
14000 rpm, to accelerate the procedure.
DNA bond to the chromato-
graphic column membrane
 Addition of AP3 buffer with ethanol and transfer into
new filter tube (natural) to allow the DNA binging
 Arrangement in centrifuge for 1 minute at the speed of
8000 rpm.

7. Washing of the DNA column
membrane
 Discrard the flowtrough and addition of AW washing buffer
(500µl) alternating 1-minute-centrifugations at the speed
of 8000 rpm, to wash the membrane and the DNA (twice).
Elution of the chromato-
graphic DNA column
 Addition of AE buffer (40 µl + 30 µl);
 Incubation for 5 minutes at room temperature;
 1-minute-centrifuge at the speed of 8000 rpm.

8. Samples reading by spectrophotomter
to measure the quantity of the extrac-
ted DNA
Once DNA is obtained, its purity is quantified and evaluated by
spectrophotometric analysis at 230,280,260 nm.
DNA concentration was evaluated by mesuring the adsorbance
at 260 nm.
The purity of DNA is based on the ratio between 230,280,260
nm assorbances:
 A260/A280 ratio allows to evaluate the contamination of
proteins (it has to be around 1,8);
 A260/A230 ratio allows to evaluate the contamination of
carbohydrates and phenols (organic solvents). It has to be
around 2,2.

9. Special thanks to CREA (Turi) and researchers: Fiammetta Ala-
gna, Maria Francesca Cardone e Carlo Bergamini.
Edited by:
Giuseppe Longo Claudia De Pascale