protocol

[Padno...@epamail.epa.gov]

I am looking for a details OP to do PCR on tail fin clips, if anyone is willing to share I would greatly appreciate it.

Beth Padnos

[Jenny Lenkowski]

On Thursday, January 10, 2013 8:52:07 AM UTC-5, Padno...@epamail.epa.gov wrote:
> I am looking for a details OP to do PCR on tail fin clips, if anyone is willing to share I would greatly appreciate it.
>
>
>
> Beth Padnos

Hi Beth,

We started using an alkaline lysis protocol that is quick and a lot cheaper than a proteinase K digestion. I have copied it below. We have also successfully used this for genotyping whole embryos, just with smaller volumes.

Best,
Jenny
Post-doctoral Fellow, Raymond Lab
University of Michigan

Alkaline Tail Lysis protocol for DNA Isolation and Genotyping
1. Into 1.5-1.7 ml centrifuge tubes, add 50 µl of Alkaline Lysis Buffer
2. Label tubes as necessary. Set a heating block to 80-95˚C.
3. Clip end of tail of adult fish and put in clearly labeled eppendorf tube, making sure that the entire tail is covered by the buffer. Be careful not to clip into the body of the fish or it will not grow back properly.
4. Place the tubes in the 80-95˚C heating block for 1 hour to digest. Vortex the tubes after ~30 minutes to break up tissue for a better digestion and to determine if longer than 1 hour are necessary for the digestion. There will be what looks like small splinters or fine hairs of tissue at this point. With fresh buffers, tails may be digested much faster than 1 hour.
5. Add 50 µl of Neutralization Buffer to each tube. Liquid can then be taken from each DNA isolation to be used directly for amplification via PCR.

Buffer/Solution Recipes
Alkaline Lysis Buffer (50 ml)
50 mg NaOH
3 mg disodium EDTA
50 ml Milli-Q
Check pH, should be near 12

Neutralization Buffer (50 ml)

315 mg Tris-HCl
50 ml Milli-Q

*best to make larger quantity than 50 ml for more accurate measurements

The buffers can be stored at room temperature, and should be remade periodically for best results (~every 6 months).