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:

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.

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.

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

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.

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.

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.

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.

Special thanks to CREA (Turi) and researchers: Fiammetta Ala-

gna, Maria Francesca Cardone e Carlo Bergamini.

Edited by:

Giuseppe Longo Claudia De Pascale