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Difficult to extract DNA from vulcanic coastal sediments


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#1 DeepSea

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Posted 03 January 2011 - 12:53 PM

Hi everyone,
i'm new writing this forum although it has been very useful in the past.
I'm trying to extract DNA from costal volcanic acidic sands preserved in RNALater with no luck. I've tried different commercial kits (MOBIO UltraClean DNA Soil kit; PowerSoil DNA Kit; Zymos DNA Kit) and manual extraction (Classic Phenol:Chloroform:IAA, EtOH precipitation; and Zhou et al 1996 extraction protocols).
All the protocols have been run in parallel with deep-sea sediment as extraction control. The deep-sea sediment (mostly fine calibrated sand and mud) worked in all trials and extracted DNA was successfully amplified by 16S PCR.
Following are some observation done on the sample (hereafter called VS - volcanic sand):

- Pellet when present are large and glossy suggesting high salt co-precipitation;
- RNAlater have been removed with three wash of the VS with 0.1 M Tris (pH 8) solution, centrifuging at 3.000 g for 10 minutes;
- Sometimes large salt cristal form even in the phenol phase during centrifugation;
- VS have been inspected under epifluorescence microscope with AO staining. cells are present in the range of 10^6-10^7 cell x g of wet/sediment weight;
- When VS is fixed with glutaraldehyde turns whitin 10 min into dark yellow color (first time I experience something like this with sediments);
- final extract have been checked for DNA presence by 0.8 % Agarose gel electrophoresis whit EtBr staining, Nanodrop 1000 spectrophotometer absorbance and 16S rDNA PCR. No bands, no absorbance and no amplification was detected with any of the former trials.

Following some consideration:

- Three 0.1 M Tris wash of sediment should remove the high salt concentration due to RNAlater
- Pellet salt have been reduced by this wash but with no DNA recovery
- Solution turn yellow with glutaraldehyde if they contain high protein concentration (Maillard-type chemistry reaction). Protein can precipitate DNA in early centrifugation steps
- Same effect could be due to high amount of metal cation in the volcanic sand

Does anybody have any suggestion on how to get this DNA extracted??

Thank you very much

D

#2 perneseblue

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Posted 03 January 2011 - 06:22 PM

For what it is worth, here are my two cents....

Try washing your sample witth 70% ethanol instead of 0.1M tris

Try using protenase K to degrade proteins in the sample..

EDTA should be able to handle divalent cations (10mM EDTA)

Glutaraldehyde reacts with amine such as proteins and tris. Glutaraldehyde also reacts with thiols and sulfides. Perhaps the glutaraldehyde reaction you are seeing is caused by sulfides?
May your PCR products be long, your protocols short and your boss on holiday

#3 DeepSea

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Posted 05 January 2011 - 08:33 AM

Hi perneseblue,
thank for your reply.
Do you mean wash the sediment before the extracion with ethanol?? I'm using 0.1 M tris wash on the sediment before to start the estraction to remove the salt from the RNAlater solution.

I will try to use proteinase K soon, at the moment i'm using lysozime, SDS and heat incubation (65?C) for the lysis.

I agree the EDTA present in the extraction buffer should be able to handle the cation.

Sulfides is present in the sediment so this is an other possible explanation...

Still no DNA from 3 to 5 grams samples extraction...




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