DNA Isolation from Reptile Blood using Gentra Puregene (Qiagen) DNA Isolation Kit

*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. ***** 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.

Turn on shaking tube incubator and set to 55°C

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 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.

Add 300µL of Cell Lysis Solution (from PureGene Kit) to each tube.

Add 10µL proteinase K (20 mg/ml) to each tube.

Close tubes and label each tube according to Sample IDs.

Add 15µL of whole blood to the correct tube and vortex well* . ***** .

Place each tube in the shaking incubator and incubate at 300rpm.

Vortex ***** the samples periodically.

Add 100µL of Protein Precipitation Solution (from PureGene Kit) to each tube and vortex ***** vigorously.

Place in ice for approximately 5 minutes.

       During this incubation do steps 12 and 13.

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).

Use a pipette to add 300µL of isopropanol into each of the newly labeled tubes.

Centrifuge the samples for 16,000x g.

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.

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.

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 the samples for 16,000x g to pellet the DNA.

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

To wash the DNA pellet, add 300µL 70% ethanol to each tube and invert several times.

Centrifuge for 16,000x g.

Again, there should be a white/clear DNA pellet visible towards the bottom of each tube.

Pour out the supernatant into waste container.

OPTIONAL: 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), add 50µL DNA Hydration Solution (PureGene Kit) and vortex* to resuspend the DNA into solution.

Incubate the tubes for one hour at room temperature, or 4°C overnight to 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).