CTAB Extraction Protocol for Sediment

THAW the CTAB-preserved sediment sample in the fridge for no more than 24h 0m 0s

DECONTAMINATE the exterior of the sample tube with 10% bleach solution and rinse with reverse osmosis water

VORTEX at max speed for 0h 0m 30s

INCUBATE at 60°C for 0h 10m 0s

ADD 15mL of Sevag (Chloroform/Isoamyl alcohol 24:1)

VORTEX the sediment/CTAB/Sevag mixture briefly

SHAKE at low speed (Vortexer setting 4) for 0h 5m 0s

CENTRIFUGE at 3220x g for 0h 15m 0s at Room temperature

CAREFULLY transfer the supernatant to a new 50 mL tube

ADD an equal volume of ice-cold Isopropanol to the supernatant

ADD 1⁄2 volume of 5M NaCl to the supernatant

INCUBATE at -20°C for 1h 0m 0s (or overnight if more convenient)

CENTRIFUGE at 3220x g for 0h 15m 0s at Room temperature

CAREFULLY pour off the supernatant

ADD 2mL of 70% EtOH, washing down the inner walls of the tube

CENTRIFUGE at 3220x g for 0h 2m 0s at Room temperature

POUR off EtOH and allow the DNA pellet to air dry completely (a 45°C incubator can be used to evaporate stubborn droplets)

RESUSPEND the pellet in 1000µL of LoTE buffer. Heat briefly at 45°C and swirl gently to mix and resuspend. Once fully resuspended, briefly centrifuge to collect all liquid in the bottom of 50-mL tube.

Transfer all liquid to a 1.5-μL low-bind microcentrifuge tube

Use 200µL in OneStepTM Inhibitor Removal Kit (Zymo Research, Irvine, CA)