Chelex extraction with safety sheet here.

FROM FISH BLOOD

Using the blood samples fixed in ethanol, 70μl of each solution was aliquoted into

sterile Eppendorf tubes (Thermo Life Sciences). Each tube was placed in a

centrifuge (MSE Micro Centuar) and pulse spun at maximum speed for 30 seconds.

Excess ethanol was taken off the top of the solution and the tubes were left open to air dry on the bench for 5-10 minutes.

After the ethanol had evaporated, 10μl proteinase K (ABgene, 20 mg/ml) and 340μl

of TEN (50mN Tris HCl pH 8.0; 100mM EDTA pH 8.0; 100mM NaCl; 1% SDS(Sodium Dodecyl Sulfate)) buffer was added to each tube. The SDS causes the cells to rupture and initiates protein denaturation while the proteinase K reduces proteins to their component amino acids. The tubes were vortexed for 30 seconds and incubated at 55oC in a rotating oven (Techne Hybridiser HB-1) for 4-6 hours.

After incubation 10μl RNAase (DNAse Free, Abgene, 2mg/ml) was added to each tube and incubated at 37oC, also in a rotating oven, for one hour in order to digest and remove traces of RNA.Following the incubation period 370μl of phenol was added to each tube to extract the denatured protein from the DNA solution. The solutions were mixed vigorously for 10 seconds and shaken gently every 5 minutes for 20 minutes.

After the 20 minute period an equal volume of chloroform was added to the tubes to absorb and eradicate traces of phenol. The solutions were again mixed vigorously for 10 seconds and then shaken gently every 5 minutes for another 20 minutes. Each tube was then spun for 5 minutes at 10,000g. This causes the solutions to separate into two distinct layers. 270μl of the top aqueous layer, which contains the DNA, was removed and pipetted into a new tube, using a wide-bore tip. 870μl of 92% ethanol (Sigma) was added to the aqueous solutions and each tube was mixed by vigorousinversions, causing the DNA to precipitate out of the solution. After 3 minutes the tubes were pulse spun (10s at 10,000g) to form a DNA pellet at the bottom of the tube.

The ethanol was decanted off and 1ml of 70% ethanol was added to each tube

to wash the pellet. Tubes were placed in a rotator overnight and the alcohol was removed using a micropipette, after which they were then left open to air dry for 10-15 minutes. The pellets were then resuspended in 100μl TE pH 8.0 (10mM Tris, 1mM EDTA) buffer.

the easiest way is to isolate DNA from just a small piece of tail fin. Then you can apply many protocols such as chelex (as another collegue mentioned above) or the salt protocol (Miller et al)

I extracted the DNA from caudal fin of tilapiine (sample wight 40-50 mg) using the DNAeasy Tissue Kit from Qiagen. It is very efficient to get a good quality of DNA as well as the quantity. I got DNA concentration approximately 400-500 ng/µl and high quality of DNA(OD 260/280 and OD 260/230 aprroximately 2 and above) when i took less supernatant (just around 150 µl) after protein precipitation. However, if you expect to get more yield, you can try using liver sample.

A very efficient method is described here: Taggart JB, Hynes RA, Prodöuhl PA, Ferguson A. 1992. A simplified protocol for routine total DNA isolation from salmonid fishes. Journal of Fish Biology 40: 963-965.

Saline extraction protocol of Sambook et al 1989

What are the most targeted growth genes in fish that have significant effects on fish muscle. What protocol can I use to extract fish growth genes?