DNA Isolation from Snake Skin Shed

Turn on shaking incubator and set to 55°C

Obtain Ice

Print list of samples

Clean the razors, scissors, and dissection boards with RNAase away; rinse with molecular-grade water.

Make fresh 70% ETOH. For example, to make 15 ml in a 15 ml tube use a 10 ml sterile pipette to take 10.5mL of 100% volume (200 proof) ETOH, and another 10 ml sterile pipette to add 4.5mL of molecular grade water.

Set out and label 1.5 mL microcentrifuge tube for each sample to use in Step 6 (Digestion).

Add 900µL Cell Lysis Buffer

Add 10µL of proteinase K( 20 mg/mL).

Set out and label a 2 mL tube for each sample to use in Step 13 (DNA precipitation).

Add containing 900µL isopropanol .

Cut up 1 in² piece of shed into smaller pieces with a sterile razor blade or scissors (change gloves and utensils between sheds).

Put shed pieces in the labeled 1.5 mL microcentrifuge tube containing the Cell Lysis Buffer and Proteinase K (prepared in Step 4).

Place in shaking incubator 300rpm Vortex occasionally for the first few hours.

Cool samples to room temperature and vortex.

Add 550µL 5M ammonium acetate to each tube and vortex for 10 seconds.

Place samples on ice for Room temperature for 10 minutes.

Centrifuge samples 17,000x g to pellet protein and debris.

Draw off as much supernatant as possible with a filtered tip into put in to a new 1.5 mL labeled tube.

Centrifuge the supernantent at second time at 20,000x g to pellet any residual protein and debris.

With a filtered tip, transfer supernatant from the second spin into the prepared 2mL tubes containing the isopropanol (prepared in Step 4).

Mix the supernatant with the isopropanol by inverting 50 times. If there is a lot of DNA you can see the strands condensing at this step (looks like thin white threads).

Place each tube into the centrifuge with the hinge facing out so the DNA pellet forms on that side of the tube. Centrifuge samples at16,000x g to pellet the DNA.

Pour off isopropanol into waste container.

Wash the DNA pellet by adding 500µL of 70% ethanol .

Centrifuge at16,000x g. The DNA pellet should still be visable.

Without dislodging the DNA pellet, carefully pour the supernatant out into waste container with as little movement as possible.

Centrifuge the tubes again for 16,000x g, and use a 10 μl tip to remove the residual ethanol. This will make the next step go faster.

Invert tubes on a paper town, with the tops open, until ethanol has completely evaporated.

Once the ethanol has evaporated (but the DNA pellet is not over dry), resuspend samples in 50µL of TE buffer .

Sit at 300rpm or leave in 4°C overnight to fully resuspend the DNA.

Run 5 µL of resuspended DNA on 1% agarose gel to visual the quality and estimate quality. DNA can be quantified with the Nanodrop. For sensitive procedures (DNA sequencing library preparation) we recommend using the Agilent TapeStation or BioAnalyzer.