TITLE: DNA Isolation from Reptile Blood using Gentra Puregene (Qiagen) DNA Isolation Kit
AUTHORS: Agl0032, tss

[DESCRIPTION]
This protocol is used to isolate DNA from reptile blood (either whole or red blood cell pellets). Reptile blood has nucleated red blood cells and will produce considerably more DNA from the same volume of mammalian blood. This protocol is adapted from Gentra Puregene DNA Isolation A Kit. 

The time estimates assumes you are processing 24 samples and you are well practiced. 

[STEPS]
SECTION: Setting Up
2. Turn on shaking tube incubator and set to 55 °C 

SECTION: Red Blood Cell Lysis
4. Set out the the blood samples to be used on ice to thaw, but stay cold. 
Set out the correct number of 1.5 mL tubes into a clean tube rack.
SECTION: Red Blood Cell Lysis
5.  Add 300 µL of Cell Lysis Solution (from PureGene Kit) to each tube.
 


SECTION: Red Blood Cell Lysis
6. Add 10 µL  proteinase K (20 mg/ml) to each tube.

SECTION: Red Blood Cell Lysis
7. Close tubes and label each tube according to Sample IDs.
SECTION: Red Blood Cell Lysis
8. Add 15 µL of whole blood to the correct tube and vortex well* . 

 


 

SECTION: Red Blood Cell Lysis
9. Place each tube in the shaking incubator and incubate at  . 
Vortex* the samples periodically.

SECTION: Isolate DNA
12. Obtain fresh 1.5 mL tubes and label top and side with DNA ID, Date, and "stock DNA" (these will be the tubes that the isolated DNA will be stored in).


SECTION: Isolate DNA
15. Without dislodging the protein pellet, pour out the supernatant containing the DNA into the newly labeled tubes containing the isopropanol. Throw away the original tube with the protein pellet.
SECTION: Isolate DNA
17. Place each tube into the centrifuge with the hinge facing out so the DNA pellet forms on that side of the tube. Centrifuge the samples for 16,000 x g, 1 min to pellet the DNA. 
 


SECTION: Isolate DNA
18. Without dislodging the DNA pellet, carefully pour the supernatant out into waste container with as little movement as possible. 
SECTION: Isolate DNA
19. To wash the DNA pellet, add 300 µL 70% ethanol to each tube and invert several times.

SECTION: Isolate DNA
20. Centrifuge for 16,000 x g, 1 min. 
Again, there should be a white/clear DNA pellet visible towards the bottom of each tube.


SECTION: Isolate DNA
21. Pour out the supernatant into waste container. 
SECTION: Resuspend the DNA
24. Once the ethanol has evaporated (but the DNA pellet is not over dry), add 50 µL DNA Hydration Solution (PureGene Kit) and vortex* to resuspend the DNA into solution. 
 

SECTION: Resuspend the DNA
25. Incubate the tubes for one hour at room temperature, or 4°C overnight to resuspend the DNA.
SECTION: Check Quality and Quantity
26. 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: Isolate DNA
10. Add 100 µL  of Protein Precipitation Solution (from PureGene Kit) to each tube and vortex* vigorously.

SECTION: Isolate DNA
11. Place in ice for approximately 5 minutes. 
           During this incubation do steps 12 and 13.
SECTION: Setting 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.

SECTION: Notice for high quality (long undamaged DNA)
1. *Through out this protocol we vortex the samples for mixing. This works well and produces  alot of good quality DNA that works for most everything. If you want to minimize damage to the DNA such as for long-read sequencing on the Nanopore or PacBIO, or assays to quantify DNA damage) do not vortex. Rather mix gently by flicking and inverting the tube. The quality will be higher but generally less yield.
SECTION: Isolate DNA
16. 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: Isolate DNA
22. OPTIONAL: 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: Isolate DNA
23. Invert tubes on a paper town, with the tops open, until ethanol has completely evaporated.
SECTION: Isolate DNA
14. Centrifuge the samples for 16,000 x g, 1 min. 
After removing the tubes from the centrifuge, there should be a yellow/brown pellet at the bottom of the tube. If there is no pellet, repeat this step for each tube missing the pellet.

SECTION: Isolate DNA
13.  Use a pipette to add 300 µL of isopropanol into each of the newly labeled tubes.