TITLE: DNA Isolation from Snake Skin Shed
AUTHORS: Agl0032, tss

[DESCRIPTION]
Purpose:
This protocol was developed for the Memphis Zoo’s Louisiana Pine Snake Breeding Project.The protocol for skin shed DNA isolation was adapted from Fetzner (1999).
 
The time estimates assumes you are processing 24 samples and you are well practiced. 

References: James W Fetzner (1999) Extracting High-Quality DNA from Shed Reptile Skins: A Simplified Method. BioTechniques 26:6

[STEPS]
SECTION: Set Up
1. Turn on shaking incubator and set to 55 °C 
Obtain Ice 
Print list of samples
SECTION: Set Up
2. Clean the razors, scissors, and dissection boards with RNAase away; rinse with molecular-grade water.
SECTION: Set Up
4. Set out and label 1.5 mL microcentrifuge tube for each sample to use in Step 6 (Digestion).
Add 900 µL Cell Lysis Buffer 
Add 10 µL of proteinase K( 20 mg/mL).

SECTION: Isolation: Lysis
7. Place in shaking incubator    Vortex occasionally for the first few hours.

SECTION: Isolation: Precipitate Proteins
9. Add 550 µL 5M ammonium acetate to each tube and vortex for 10 seconds.

SECTION: Isolation: Lysis
8. Cool samples to room temperature and vortex.
SECTION: Isolation: Precipitate Proteins
10. Place samples on ice for Room temperature for 10 minutes.

SECTION: Isolation: Precipitate Proteins
11. Centrifuge samples 17,000 x g, 5 min to pellet protein and debris.

SECTION: Isolation: Precipitate Proteins
12. Draw off as much supernatant as possible with a filtered tip into put in to a new 1.5 mL labeled tube.
SECTION: Isolation: Precipitate Proteins
13. Centrifuge the supernantent at second time at 20,000 x g, 3 min to pellet any residual protein and debris.

SECTION: Isolation: Precipitate DNA
14. With a filtered tip, transfer supernatant from the second spin into the prepared 2mL tubes containing the isopropanol (prepared in Step 4). 
SECTION: Isolation: Precipitate DNA
16. Place each tube into the centrifuge with the hinge facing out so the DNA pellet forms on that side of the tube.  Centrifuge samples at16,000 x g, 2 min to pellet the DNA.
 

SECTION: Isolation: Precipitate DNA
17. Pour off isopropanol into waste container.
SECTION: Isolation: Precipitate DNA
18. Wash the DNA pellet by adding 500 µL of 70% ethanol.

SECTION: Isolation: Precipitate DNA
19. Centrifuge at16,000 x g, 2 min. The DNA pellet should still be visable.

SECTION: Isolation: Precipitate DNA
20. Without dislodging the DNA pellet, carefully pour the supernatant out into waste container with as little movement as possible. 
SECTION: Isolation: Precipitate DNA
21. Centrifuge the tubes again for 16,000 x g, 1 min, and use a 10 μl tip to remove the residual ethanol. This will make the next step go faster.
SECTION: Isolation: Precipitate DNA
22. Invert tubes on a paper town, with the tops open, until ethanol has completely evaporated.
SECTION: Resuspension
23. Once the ethanol has evaporated (but the DNA pellet is not over dry), resuspend samples in 50 µL of TE buffer. 

 


SECTION: Check Quality
25. 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.
SECTION: Set Up
5. Set out and label a 2 mL tube for each sample to use in Step 13 (DNA precipitation).
Add containing 900 µL isopropanol.

SECTION: Isolation: Lysis
6. 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).
SECTION: Isolation: Precipitate DNA
15. 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).
SECTION: Resuspension
24. Sit at   or leave in 4°C overnight to fully resuspend the DNA.

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