DNAzol method

FTA PlantSaver card method

Materials: Fresh and healthy leaf sample (15 - 21 day old); Mortar and pestle; DNAZOL reagent; Absolute ethanol; 70% ethanol; DNAzol-ethanol wash (optional); Chloroform timer; Micropipettes; 1.5 ml microcentrifuge tubes; Vortex machine; Centrifuge machine; Freezer, plastic rack, hand gloves and lab coat.

Materials: Fresh and healthy leaf sample (15 - 21 day old); FTA PlantSaver card; PestleParafilm paper; Harris punch; Harris cutting mat; Cotton wool; Desiccator; Absolute ethanol; FTA purification reagent; 70% ethanol; Timer; Micropipettes; 1.5 ml microcentrifuge tubes; Vortex machine; Plastic rack; Freezer; Hand gloves and lab coat.

Label your microcentrifuge tube (1.5 ml - 2 ml capacity) with a number representing the accession code.

Place a leaf sample in a labeled square of the FTA card.

Weigh 1 g of leaf sample and place in a mortar. Add 5 ml of absolute ethanol to submerge the leaf tissue for 30 minutes. Decant excess ethanol.

Overlay the sample with a transparent parafilm.

Dispense 750 µl DNAzol reagent into the tube

Gently pound the leaf until greenish sap is transferred beneath the paper.

Pulverize the leaf tissue in the mortar.

Remove the parafilm and air dry the card for 1 hour.

Transfer the homogenize tissue to the tube containing DNAzol. Allow the mixture stand for 5 minutes.

Cut 2 discs (2 mm diameter) from the sample into a 1.5 ml tube using the Harris punch.

Add 750 µl chloroform to the mixture above. Allow the mixture stand for 5 minutes.

Add 200 µl of 70% ethanol to the tube and allow soaking for 5 minutes. Vortex for 30 minutes before discarding the liquid, leaving the discs in the tube.

Centrifuge the tube at 10,000× g for 10 minutes.

Repeat the last step.

Transfer the supernatant (portion containing the DNA) into a new and labeled tube.

Dispense 200 µl of FTA purification reagent to the tube; allow to soak for 5 minutes. Vortex for 30 minutes before discarding the liquid gently, leaving the discs in the tube.

Add 750 µl absolute ethanol to the transferred supernatant to precipitate the DNA for 5 minutes.

Repeat the last step.

Centrifuge at 5000× g for 5 minutes to produce pelletized DNA.

Transfer the two discs containing purified DNA into a fresh tube using micropipette tips.

Add 750 µl of 70% ethanol to resuspend the pelletized DNA. Allow the mixture to stand for 5 minutes.

Air the discs in the tube for 1 hour.

Further centrifuge at 5000× g for 5 minutes.

Store in a freezer at −20˚C for further use.

Gently decant the liquid portion leaving the pelletized and pure DNA extracted.

Each disc can serve as a DNA template for PCR.

Air dry the tube for 1 hour. Reconstitute the DNA in 100 µl 1× TE for further use.

Comment: FTA method is recommended for timely and quality DNA extraction and amplification from large number of samples.