CTAB DNA Extraction Protocol for Mollusks

Slice tissue sample using a clean, sterile scalpel. Put sample into a 1.5 ml tube with 400µL .

Add 10µL 20mg/mL and invert tube to mix.

Incubate at 50°C for 60 minutes until lysed (or longer depending on tissue thickness). Invert tube occasionally to mix sample.

Add 400µL . Gently shake tube to emulsify sample.

Leave at room temperature for 2 minutes and shake tube to mix.

Spin tube at 13 000 rpm for 2 minutes and transfer upper layer to a 1.5 ml tube.

Add 5µL of 10mg/mL . Leave for 30 minutes at 37°C .

Add 1 equal volume of .

Spin tube at 13 000 rpm for 2 minutes and transfer upper layer to a new 1.5 ml tube containing 900µL CTAB dilution solution . Gently invert tube to mix. Leave in fridge overnight (optional) .

Spin tube at 13 000 rpm for 10 minutes.

Discard supernatant with a micropipette and add 1000µL 0.4 M NaCl in TE . Invert tube to wash.

Spin tube at 13 000 rpm for 5 minutes and remove supernatant.

Add 300µL 1.42 M NaCl in TE to dissociate the CTAB from the DNA. Invert tube until pellet becomes transparent.

Add 600µL (previously left at -20°C ). Invert manually until precipitation appears.

Centrifuge at full speed for 8 minutes, then remove supernatant. Dry at room temperature or in heat block at 37°C

Resuspend in 50µL EB buffer. Use lesser volume of EB buffer if a higher concentration is required.

All centrifugations were performed using a benchtop centrifuge (Eppendorf - 5415 D)

The Nucleic acid quality is measured using a Nanodrop, and the A260/A230 ratio of 1.8-2.0 indicates good DNA purity. Qubit can be used to determine nucleic acid concentration.

Store DNA sample at -20°C until further use.

CTAB Extraction Solution

CTAB Dilution Solution

0.4M Nacl in TE

1.42 M NaCl in TE