Root Surface Sterilization for Plating and Fungal DNA isolation
1. Cleanse roots by rinsing in water in a plastic petri dish. Remove as much soil as
possible. Select random roots and make sure you have a good representative
sample.
2. Chop roots into 1 cm fragments with a surface sterilized (alcohol flamed) razor
blade, small scissors or scalpel. You should have a minimum of 10 and up to 30
1-cm segments. Select roots with fine root hairs if they are available. Make sure
you get a little of everything: small and thick roots, root segments from each end
of the root and the middle.
3. For the following steps, Time is critical! If you leave the root fragments in
the bleach or ethanol too long the fungi in the roots will be killed. Remove any
excess water from the petri dish and add 70% ethanol for 1 min. Pour off the
ethanol and add 1% bleach (sodium hypochlorite) for 1 minute. Pour off the
bleach and rinse three times with sterile water. To discard the liquid (water,
bleach or ethanol), tilt the plate vertically with the lid closed to avoid losing root
pieces. After the last wash pour off excess water.
4. Transfer all but 5 of your fragments into a small Ziploc bag. These will be put in
the freezer until we are ready to isolate DNA. Be sure this bag is labeled with
your sample name and your initials
5. Place the remaining 5 fragments on an MEA/ampicillin plate using alcohol
flamed sterile forceps. Label the plate with your sample name and your initials.
Solutions
Sterile water
70% ethanol
Bleach diluted to 1% sodium hypchlorite (note: commercial bleach is usually in the order
of 8% sodium hypochlorite)