DNA extraction from spruce seed coat - (Feb/09/2006 )

Hi!

I have problems extracting DNA from spruce seed coat & wings. I only have seed samples and I need maternal genotype thus extraction from embryo in out of the question.
After crushing material in liquid N2 I have used standard 2% CTAB protocole with 1%PVP and chloroform :IAA (Doyle&Doyle) and DNeasy mini kit (Quiagen). No DNA was visible on 1% agarose gels (5µl DNA + 2µl loading buffer) and there was no PCR product either. I have used the same extraction protocols and PCR primer pairs for fresh buds before and they have worked fine.
I think the problem could be starting material (it has been stored at room temperature since 2002). Does anybody have experience with so small amounts (one seed coat weighs ca 0,1g) of (presumably) degraded starting material? What should I do to make my extractions work?
I'd appreciate any idea or suggestion. Thanks.

Are you shore that the seed coat is a living tisue?
If not that's why you'r not geting anything from it.

Alternatively try the methods from:
http://www.ejbiotechnology.info/content/vo...e1/full/3/3.pdf

Tahnks for the protocol. I'll try it out.

I have read in a paper by Ziegenhagen et al. (2003) that CTAB DNA extraction was possible from oak pericarp (the wall on the oak seed) and from silver fir seed wings. They report yield of 365 ng/ul for oak pericarp and for fir seed wing 41 ng/ul. Therefore I assume there should also be enough DNA in seed coat and seed wing of Norway spruce (I have tried to extract DNA from the latter also but with no success).

Hi

I have successfully extracted DNA from seed coat of Brassica napus, and have obtaind good results for PCR too. I have extracted DNA using Qiagen Mini Plant extraction kit. It is easy and simple and one gets good amount of DNA. If you have any question please ask.
Best of luck.
Kunal.