RE [Cnidaria] Genomic DNA

[Peter.Schuchert at ville-ge.ch]

> Would anyone be willing to share a nice-quick protocol  or (in your
> experience) a commercial kit’s name that best suit for genomic
> isolation in jellies.


I use the cTAB protocol, which works very well and gives good yields. For
very small medusae I have also used Qiagen DNeasy columns. They give good
quality DNA, but the yield is normally very poor.

cheers,
Peter


CTAB protocol for living or alcohol preserved tissues:
1.    add CTAB 1x buffer  (normally 200 μl)
2.    add Proteinase K 10 mg/ml, normally 10 μl (avoid too high
concentrations)
3.    55°C for at least 5 h, up to 24 h possible.
4.    add same volume of chloroform+isoamylalcohol (=3-Methyl-1-butanol)
24+1, vortex vigorously
5.    centrifuge 5 min. max. rpm
6.    upper phase in new eppentube, avoid any contamination with the
interphase or lower phase
7.    add 2 volumes of ethanol 100%, mix
8.    10 min centrifugation at max. rpm
9.    pour out liquid, gently add 0.5-0.7 ml 70 % ethanol, pour out and let
drain on tissue
10.   dry DNA at 55°C 5min
11.   dissolve in 50-200 μl TE buffer

put 2 to 8 μl on gel, estimate approximate yield by comparing with DNA
sample of known concentration.


Buffer 1x CTAB: 07 M NaCl, 10 mM EDTA, 50 mM Tris pH 8.0, 2%
       hexadecyltrimethylammonium bromide CTAB