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. |