Thank you dear Arthur for the articles. Still has there been any instances of successful application of phenol-chloroform method?

well my access to kits is a bit limited for a couple of months and so I am reserving them for the problematic possible individuals. So until then I figured out I would use the traditional method. Plus I am planning to have a workshop on DNA extraction for some other students based on the method

Do you believe CTAB is gonna work?!! cause as the history of it shows it has been dominantly used for plants

Hi, we are routinely using regular phenol-chloroform protocol in our lab and it works perfectly for amphipods preserved in 96% ethanol, even for a very tiny piece of tissue (a piece of a muscle, one gnathopod etc.). Even if we don't see too much of a pellet at the end of extraction, the amount of DNA is perfectly OK for PCR of both nuclear and mitochondrial markers.

I have also used CTAB based protocol for other crustaceans (mainly penaeid shrimps) and it was also working OK.

Have you actually tested your extracted DNA for PCR?

Best greetings,

Michal

Hi Michal

actually due to some reason I can't seem to get my PCR to work though my DNA concentration measured through NanoDrop is about 1.5 ug/ul which seems to be fine to the PCR. I have tried Hou et. al 2007 PCR protocol for 16S and CO1 and yet nothing. 

What do you think is the reason?

Best

Ahmad-Reza

That is strange, we are also working (among others) with the PCR protocols from Hou et al. 2007 and their other papers. I think we should concentrate on the way the samples are preserved: what kind of ethanol, pure or contaminated, how old are the samples, how they are kept in the collection etc. As for your DNA concentration - I think that it may be too big for the PCR - I would make a series of dillutions down to 10-15x and check if that helps.

Best greetings,

Michal

Dear Michal

My samples were transferred directly to 96% ethanol and kept in 4 degrees C. 

Is there any thing else I should suspect?

Ahmad-Reza

This seems an ideal treatment... I would try the series of dillutions now.

Best,

Michal

Do you have any exact suggestions Michal? I have tried 5 ul of my DNA instead of 1

Best

Ahmad-Reza

In my opinion you have to reduce the amount of the DNA by dilluting your matrix DNA by, let's say, 5, 10 and 15x. Then try to make the PCR on each of the dillutions. That may help.