How to Extract DNA From Plant Leaves

Fill the waterbath to the recommended level with water, which varies with each manufacturer's specifications, and turn it on. Set the waterbath to 65 degrees Celsius and allow it to come up to temperature. Place the isopropanol container on ice. Set the centrifuge at 3,000 revolutions per minute at a temperature of 20 degrees Celsius.

Weigh 2 g leaves on a balance and place them into a mortar. Add enough beta-mercaptoethanol via pipette to a container of extraction buffer so the final product is made up of one to two percent of beta-mercaptoethanol. For example, if you have a volume of buffer that measures about 100ml, then add 2 ml beta-mercaptoethanol to the container and mix thoroughly.

Pipette 7 ml extraction buffer into the mortar. Crush the mixture into a homogeneous liquid with the pestle.

Fold a piece of cheesecloth over to form four layers. Strain and squeeze the liquid through the layers into a 15-ml centrifuge tube.

Place the tube into the waterbath for 30to 60 min utes. Swirl the tube to mix its contents every quarter of an hour. Allow the tube to cool to room temperature after the full time period is up.

Top up the tube with the chloroform and isoamylalcohol mixture (this contains 24 parts chloroform to one part isoamylalcohol), so the tube contains roughly half the sample and half the new liquid addition. Cap the tube and turn it upside down a few times slowly so the fluid mixes.

Put the tube in the centrifuge and let it spin for 10 minutes.

Place 50 ml RNAse A into a new centrifuge tube. Pipette the supernatant (the clear layer at the top of the tube above a white layer of debris) in the first tube off and move it to the second tube. Cap the tube and invert it a few times to mix the contents together. Keep the tube at room temperature for an hour.

Perform steps 6 and 7 over again twice so you have a clear sample tube. Then pipette up to 9 ml of the clear liquid from the final tube into a new tube. Add 6 ml isopropanol and invert the tubes 10 times in a gentle manner so the DNA falls out of solution and into a solid form. Then centrifuge the tube for 10 minutes, which should create a pellet of DNA in the bottom of the tube.

Pour off the liquid in the tube so the pellet remains. Pipette in 2 ml 70% ethanol and place back in the centrifuge for five minutes. Pour off the liquid portion again. Use a sterile pipette tip to pick up the pellet and move it to a sterile 1.5-ml Eppendorf tube. Pipette in 200 microliters of sterile deionized water and let the DNA dissolve entirely in the liquid, which can take overnight. The sample is then ready for analysis.