The potassium acetate is by far the best. As a matter of fact I routinely use it in all my DNA/RNA extractions as an added step for cleanup whether eukaryotic or procaryotic. Therefore I would suggest the following:

1. Use whatever crude extraction method gives you the best yield (not purity).

2. After the above precipitation of nucleic acid (and everything else), res-suspend in a generous amount of TE and then add an equal volume of GHCL buffer buffer [6.5M guanidinium hydrochloride, 100mM Tris-HCl (pH 8.0), 0.1M sodium acetate (pH 5.5), 0.1 M β-mercaptoethanol, 0.2M KOAc] . Spin down to selectively precipitate contaminants including polysaccharides.

3. Do Phenol/chloroform extraction and precipitate.

If problem persist I would follow up the above with aliginase, but I am very much doubtfull. Also the above GHCL buffer is compatible with Qiagen collumns, simply load (after the spin) and wash with the manufacture's wash buffer. I used the above buffer to extract RNA from abalone (Pollysaccharide rich) and then cleanup with Qiagen RNeasy collumns. It was the only time in my life that I actually got the maximum yield as per binding capacity in the manual which is indicative of minimal contaminants. Also see the below reference,

Regards

Marnus

Ps are you sure about your choice of next generation sequencing platform

Dos Reis Falcao, V., A. Pedroso Tonon, et al. (2008). "RNA isolation method for polysaccharide rich algae: Agar producing Gracilaria tenuistipitata (Rhodophyta)." Journal of Applied Phycology 20(1): 9-12.

The potassium acetate is by far the best. As a matter of fact I routinely use it in all my DNA/RNA extractions as an added step for cleanup whether eukaryotic or procaryotic. Therefore I would suggest the following:

1. Use whatever crude extraction method gives you the best yield (not purity).

2. After the above precipitation of nucleic acid (and everything else), res-suspend in a generous amount of TE and then add an equal volume of GHCL buffer buffer [6.5M guanidinium hydrochloride, 100mM Tris-HCl (pH 8.0), 0.1M sodium acetate (pH 5.5), 0.1 M β-mercaptoethanol, 0.2M KOAc] . Spin down to selectively precipitate contaminants including polysaccharides.

3. Do Phenol/chloroform extraction and precipitate.

If problem persist I would follow up the above with aliginase, but I am very much doubtfull. Also the above GHCL buffer is compatible with Qiagen collumns, simply load (after the spin) and wash with the manufacture's wash buffer. I used the above buffer to extract RNA from abalone (Pollysaccharide rich) and then cleanup with Qiagen RNeasy collumns. It was the only time in my life that I actually got the maximum yield as per binding capacity in the manual which is indicative of minimal contaminants. Also see the below reference,

Regards

Marnus

Ps are you sure about your choice of next generation sequencing platform

Dos Reis Falcao, V., A. Pedroso Tonon, et al. (2008). "RNA isolation method for polysaccharide rich algae: Agar producing Gracilaria tenuistipitata (Rhodophyta)." Journal of Applied Phycology 20(1): 9-12.

One way that work for us in grapes (to much polysaccharides at mature stage), was to work with the Modified CTAB method (Reid, K., Olsson, N., Schlosser, J., Peng, F. and Lund, S. (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biology 6: 27.)...basically the buffer is prepared with 300 mM of Tris HCl (pH 8) instead of 100 mM of the regular CTAB Buffer, plus the 30.000 x g centrifugation step........ we added a additional Phenol/chloroform extraction step.........

****Important: remember that the protocol is designed for RNA isolation, so, don't add spermidine to the CTAB buffer for DNA isolation*****.

I agree with Marnus about the clean up with Qiagen RNeasy collums, too.

Cheers,

Felipe Gainza.

It sounds like you're doing a lot of home-brew extraction methods, in which case Marnus has some great suggestions. Potassium acetate is great for removing not just the polysaccharides but the proteins as well. However, those precipitation steps can be time consuming and result in a lower yield as each precipitation step reduces the total amount of DNA recovered. As Felipe mentioned, CTAB is traditionally for RNA, but should get the total nucleic acids. If you can afford it, I suggest that you put the extracted DNA on a column for PCR clean-up such as Zymo clean and Concentrator or Omega PCR Clean Up - this will bind the DNA in high salt, wash away the contaminants in ethanol and elute in water or 10mM tris. These are traditionally cheaper than DNA extraction kits provided by Qiagen and MoBio and literally take only 10 minutes to clean up as it only requires 4X 30second spins in a centrifuge.

We experienced similar problems with Ectocarpus samples. The PCR clean-up columns did not make any difference. We got the best results combining the extraction protocol of Pearson adapted for DNA (the ethanol polysaccharide precipitation step is very important) and incubation of re-suspended DNA with Chelex 100 solution.

Try the following:

• Make 10% Chelex 100 solution in HPLC water.

• Vortex solution before every use (resin sediments on the bottom of the tube)

• Mix 180μl of 10% Chelex with 20μl of DNA sample (gDNA, cDNA, etc.) You can use ng to μg nucleic acid.

• Incubate at 56⁰C for 20 minutes  vortex sample after 10 minutes.

• Centrifuge for 3 minutes at 12000g to spin down the resin.

• Transfer the liquid to a fresh tube, be careful not to transfer any Chelex! It can interfere with downstream applications.

Regards,

Agnieszka

[1]Pearson G, Lago-Leston A, Valente M, Serrão E: Simple and rapid RNA extraction from freeze-dried tissue of brown algae and seagrasses. European Journal of Phycology 2006, 41:97–104.