DNA extraction for mate pair lib. from pigmented marine inverts

[kmkocot]

Hi all,

I need to extract DNA from some frustrating marine invertebrates. They are slimy and have a pigment that must have some chemical properties similar to DNA. When I do an extraction with phenol/chloroform, the Qiagen DNeasy kit, or even the MoBio PowerSoil kit, the resulting DNA is dark brown when resuspended in water. Nanodrop can't make heads or tails of it. I'm fairly limited on material (this is a deep sea, Antarctic species) and I need about 50 micrograms of high molecular weight DNA for an Illumina mate pair library. I was wondering if you have any extraction or re-precipitation suggestions. Maybe Ampure beads?

Thanks!
Kevin

[nucacidhunter]

With all those methods that you have described, AMPure seems to be the best product. In theory DNA should precipitate and anything else will be washed off. You can use 1.6x bead and first try a small portion of your extracted DNA. You need dsDNA PicoGreen (if it works with the pigment in your DNA solution) to quantitate before and after clean-up to make sure you are not losing your DNA (10-20% loss is normal). Sometimes pigments do not interfere with downstream application and you can try to prepare a small scale library to see if the colour adversely affects you prep.

[kmkocot]

Thanks for the input. I neglected to mention this stuff definitely inhibits PCR (DNA has to be diluted to absurdly low concentrations for PCR to work) so I'm guessing it would cause problems in library prep as well.

Thanks!
Kevin

[nucacidhunter]

I neglected to mention this stuff definitely inhibits PCR (DNA has to be diluted to absurdly low concentrations for PCR to work) so I'm guessing it would cause problems in library prep as well.

It depends on the platform and the mate pair kit you are going to use. There will be many clean ups during library prep as well, so gradually it may get cleaned and not to cause much problem by the time it gets to PCR step. Inhibiting DNA polymerase does not always translate to inhibition of other enzymes such as ligases because they catalyse reaction with different mechanisms.

[GW_OK]

Yeah, in another life I had severe issues with plant DNA/RNA having brown polyphenols acting as PCR inhibitors. I'd try the ampure beads for sure. Also, I'd not use a nanodrop at all for quantification. Get a Qubit or some other fluorometer (not that it would be immune to polyphenolic contaminants...)