Protein contamination in bottle gourd DNA extraction

[logan]

Hey everyone,
I've been using a CTAB extraction protocol to isolate DNA suitable for PCR from single caryophillid seeds for a while now, and I recently tried the same with bottle gourd (Lagenaria siceraria, Cucurbitaceae). With the Caryophillids, I incubate the single seed in CTAB buffer for one hour, grind with pellet pestle, and proceed as normal, no liquid nitrogen is used. With bottle gourds, I've been excising a small amount of embryo tissue and attempting the same procedure, but I'm ending up with very low 260/280 values. I added proteinase K along with CTAB buffer in the last attempt, but no luck. Any suggestions? Thanks very much.

Logan

[JackBean]

I guess you are not using only the CTAB buffer, are you? So, writte you consequent steps, there must be the answer...
(the step, where you precipitate proteins and remove them or where you precipitate DNA)

[logan]

1) Incubate at 55C in 500 uL CTAB buffer including 0.02g PVP and 2.5 uL mercaptoethanol.
2) Grind tissue directly in buffer, add chloroform, centrifuge, recover upper phase.
3) Incubate on ice with ammonium acetate and isopropanol. spin down and pour away supernatant.
4) Wash pellet twice with ethanol and resuspend in TE buffer.

I read it's possible after DNA precipitation to add distilled water and re-precipitate proteins in 5 M ammonium acetate, so I'll give that a try. Apparently this taxon has caused other people similar problems, but I'm having issues running down exact fixes just yet.