1. DNA Preparation
<ol>
	<li>Prepare DNA lysis buffer: 50 mM KCl, 10 mM Tris-HCl pH 8.3, 0.3% Tween 20, 0.3% NP40. Add fresh Proteinase K to a final concentration of 1 mg/ml on the day of use.</li>
	<li>Tissue collection:
	<ol>
		<li>For adult fish fin clip:
		<ol>
			<li>Anesthetize fish: Place the fish in 0.004% MS-222 (tricaine) solution. Wait until gill movement slows.</li>
			<li>Put fish on a stack of 5-10 Kimwipes and cut a small piece of the tail fin, about 2-3 mm, with a sterile razor blade.</li>
			<li>Quickly place the fish in a labeled tank with fresh water for recovery; carefully label both the tank and corresponding tube that will contain the fin clip. Change the water and feed the fish every other day.</li>
			<li>Pick up the fin clip with a sterile pipette tip and transfer it into a tube filled with 100 &#956;l DNA lysis buffer.</li>
			<li>Discard the pipette tip, and discard the razor blade into a sharps container.</li>
		</ol>
		</li>
		<li>For embryo tail clip:
		<ol>
			<li>Place embryos into 0.004% MS-222 (tricaine) solution. Wait 2 min.</li>
			<li>Cut a piece of tail with forceps under a dissecting microscope.</li>
			<li>Put the tail into a labeled tube filled with 30 &#956;l DNA lysis buffer.</li>
			<li>Quickly put the embryo into a tube containing 100 &#956;l 4% paraformaldehyde.</li>
			<li>Label tubes with embryos and their corresponding tail tubes.</li>
			<li>Store the tubes with embryos at 4 &#176;C overnight for fixation.</li>
			<li>Clean the forceps using Milli-Q water and Kimwipes between each embryo to minimize contamination.</li>
		</ol>
		</li>
		<li>For whole embryos:
		<ol>
			<li>Place embryos into 0.004% MS-222 (tricaine) solution. Wait 2 min.</li>
			<li>Put 1-5 embryos in a tube filled with 100 &#956;l DNA lysis buffer. Dechorionation is not necessary.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>DNA digestion 
	Incubate tubes with tissue (fin or tail clips or embryos) at 55 &#176;C for 4 hr up to overnight.</li>
	<li>Inactivate the Proteinase K 
	Heat tubes to 95 &#176;C for 15 min. DNA should be used for PCR immediately, or stored at -20 &#176;C for up to 3 months.</li>
</ol>

<table><tbody><tr><td>100 Reaction LightScanner Master Mix</td><td>BioFire</td><td>HRLS-ASY-0002</td><td>www.biofiredx.com</td></tr><tr><td>Tricaine&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td></td><td></td><td></td></tr></tbody></table>

dna extraction from tail clip: a method used in zebrafish genotyping

DNA Extraction from Tail Clip: A Method Used in Zebrafish Genotyping

Source: Xing L., et al. <a href="https://www.jove.com/v/51138/" target="_blank">Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis</a>. J. Vis. Exp. (2014).
This article describes DNA extraction from zebrafish tail clip. The protocol demonstrates a rapid and efficient method to analyze zebrafish genotypes.

Source: Xing L., et al. Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis. J. Vis. Exp. (2014).
This article describes ...

- Start the procedure by placing an anesthetized adult fish on a stack of absorbent paper. Now use a sterile razor blade to cut the tail fin and quickly transfer the fish to a tank containing fresh water for recovery. Transfer the tail clip to a clean tube containing a lysis buffer. The lysis buffer contains nonionic detergents, which solubilize the plasma membrane and the nuclear membrane of a cell. The buffer also contains an enzyme called proteinase K that breaks down proteins and enables the release of DNA into the lysate. It also degrades nucleases, protecting the DNA from nuclease digestion.
Now, leave the tubes with tail clips in an incubator overnight to completely break down the tissue at 55 degrees Celsius with continuous rotation. Next, heat the tube at 95 degrees Celsius for 15 minutes to inactivate proteinase K. Centrifuge the tube to pellet undigested material and transfer the supernatant containing DNA to another tube. Add alcohol to precipitate the DNA and centrifuge again to collect the DNA in a pellet. In the following protocol, we will isolate DNA from the tail clip of an adult zebrafish and from a whole embryo.
- On the day of the DNA preparation, add fresh prot

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JoVE Encyclopedia of Experiments

Biology

Danio rerio (zebrafish)

Adult

6.5K Views. - Start the procedure by placing an anesthetized adult fish on a stack of absorbent paper. Now use...

Abstract

Protocol

<table><tbody><tr><td>100 Reaction LightScanner Master Mix</td><td>BioFire</td><td>HRLS-ASY-0002</td><td>www.biofiredx.com</td></tr><tr><td>Tricaine&nbsp;</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td></td><td></td><td></td></tr></tbody></table>

DNA Extraction from Tail Clip: A Method Used in Zebrafish Genotyping

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Abstract

Protocol

Materials

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