DNA Extractions using CTAB
From the laboratory of the Institute for Genomic Diversity at Cornell University

• LABEL 2 sets of costar tubes
• PUT METAL BALLS in one set.
• COLLECT leaf tissue, about 0.17g per tube. (Remove the midrib)
• While harvesting keep rack on ice and move to -80C when rack is done.
• If only extracting one plate, divide samples onto two plates
• Keep samples at -80C overnight until ready to grind.
• Make sure there is isopropanol in the freezer.

Next Day (always keep any open tubes in hood):

• Turn on 60C water bath.
• Prepare all solutions and ready the hood.
• MIX 120ul B-mercaptoethanol with 60ml CTAB (0.2% B-mercaptoethanol)
• Prepare a multi-channel pipette (8 x 250 ul with tips) and plastic trough
• FILL the bottom of a Styrofoam box with liquid N₂.
• GENOGRIND samples at 500 strokes/min for 20 sec.
• If not fully ground, put tubes on liquid N₂ to refreeze
• Genogrind another 20 sec.
• Repeat a third time if necessary.
• ADD quickly- 250 ul of CTAB/B-mercaptoethanol to all samples, then add another 250ul (500 ul total).
• PLACE all capped tubes in 60C WATER BATH, 5 min.
• INVERT: Make sure caps are on tightly, put paper towel between tubes and lid, and invert until no tissue remains stuck to the bottom of tubes.
• PLACE AT 60C water bath FOR ANTOHER 10 min.
• Wear 2 layers of blue nitrile gloves or the thick green gloves
• Pour CIA (chloroform-isoamylalcohol) into 1L GLASS beaker
• ADD 200 ul CIA to all samples, then add another 200 ul CIA (400ul total).
• INVERT: Cap tightly, place paper towel between cap and lid and invert 50-70 times under the hood.
• SPIN down: speed 5600, 5 min (in BRC).
• TRANSFER SUPERNATANT to clean tubes (double gloves)
• ADD 300 ul of ISOPROPANOL (from freezer)
• Cap carefully and INVERT until mixed (about 15 times)
• PLACE in FREEZER for 1 hour or overnight
• SPIN down: speed 5600, 5 min.
• DECANT
• ADD 300 ul 70% EtOH (from freezer).
• Put in freezer 15 min or overnight
• SPIN down: speed 5600, 2 min.
• DECANT
• ADD 300ul 90% + EtOH (from freezer).
• PLACE in FREEZOR15 min or overnight
• SPIN down: speed 5600, 2 min.
• REMOVE ETOH with pipette: draw off as much as you can without sucking up the pellet.
• LET DRY in hood, but don’t let them dry out entirely.
• RESUSPEND in 100 ul TE (if small pellet then can resuspend in less)
• If pellets don’t resuspend after 15 min at room temp, place in 40C (or cooler) water bath with caps off for 5 min MAX then vortex gently.