We are having problems in PCR amplification of DNA extracted from fish eggs (tuna). DNA extracted from muscle tissue using the same methodolgy works fine. Any help would be really appreciated.
There are several protocols available online that are specific for DNA extraction from fish eggs for PCR. Could you be more specific as to what extraction method, or problem that you are having? Is it with the extraction, or with the PCR.
Have you tried PCR on the fish eggs with primers that should definitely work - example RNA genes?
Thank for the answers. We tried a modified phenol chloroform method and also commercial DNa isolation kit. In both cases, the DNA seems ok, and mtDNA markers works perfectly. but we still having problems with the amplification of the micro satellites
I would rather suggest you to isolate DNA using a commercial DNA isolation kit or purify the DNA samples using columns. Though the phenol:chloroform method yields good quality of DNA, sometimes traces of phenol creates problem in PCR amplification. Good luck.
If you don't want spend money on commercial DNA isolation kit, try the salt extraction. It is similar methodology compare to phenol extraction and should give better result. I add a reference below:
http://nar.oxfordjournals.org/content/25/22/4692.long
PS: Did you test the same microsatellite markers on muscle tissus ?
My suggestions would be:
1) start up with a good extraction method, which you can modify to avoid washing up too much DNA. We are using Genomed JETquick and it works well. It works on pipefish eggs and body mucous. (See Monteiro NM, Silva RM, Cunha M, Antunes A, Jones AG, Vieira MN. 2013. Validating the use of colouration patterns for individual recognition in the worm pipefish using a novel set of microsatellite markers. Mol Ecol Resour).
2) Obviously, it is worth using a good taq. We are having results with platinum from Invitrogen that were not possible with other taqs of lesser quality (especially in situations such as these were DNA does not abound).
3) It is worth considering also how long were the eggs fertilised. If they are "freshly" fecundated, then you might not pick up a signal of your microsatellites. Nevertheless, we are picking up signals from very early on of the embryonic development.
So, not sure if any of this helps, but If you want to discuss the protocols that we are using, fell free to mail.
Cheers,
Nuno
If there were problems with the extraction method that affected the DNA, then you should not get results using the mitochondrial primers. Since you are getting results, DNA quality should not be an issue.
This sounds like a template concentration problem. Have you tried PCRs on a series of concentrations of DNA? Start with what you think would be way too concentrated, and continue over a range of concentrations. Would be good to do this in conjunction with a dilution series of the muscle DNA.
Did you resolve your problem with amplification of microsatellites from tuna eggs?
One crucial step that improved our DNA extraction protocol from fish eggs was to rinse the egg in water (if stored in alcohol) and then use a sterile tip to burst the egg inside the extraction tube prior to the next step. I hope this helps. Luca
Luca. Thank you for your answer. We are now having amplification at not very high rates. We will try to burst the egg.
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