After precipitation of DNA with ethanol and sodium acetate, there are many crystalline precipitates with the DNA pellet. I suppose sodium acetate. NanoDrop show a peak at 230 nm.
My protocol:
500 µl DNA in a.dest + 1/10 vol. NaOAc (3M, pH 5.2) + 2.5 vol. abs. EtOH, 30 min at -80°C centrifugation at 14.000 g, 4°C, 30 min. washing with 70% EtOH, centrifugation at 14.000 g, 4°C, 10 min.
How can I avoid salt-precipitates?
Hi, Daniel,
I don't know what the crystalline precipitates are. I doubt they're sodium acetate crystals because NaAc is very soluble. I believe it's an unpurified contaminant inherent to the samples you're purifying.
I see the 230 peak in my samples on the Nanodrop after purifying DNA plugs from agarose gels. This is residual guanidine. The NanoDrop manual (link below) also says the 230 peak can be:
• Carbohydrate carryover (often a problem with plants).
• Residual phenol from nucleic acid extraction.
• Residual guanidine (often used in column based kits).
• Glycogen used for precipitation.
You can remove these impurities with an ethanol precipitation or another round using the kit, although this will result in lower yields.
I use my samples containing the 230nm peak in downstream reactions like ligations and PCR without much trouble (depends on the peak height). I would recommend you do the same but the precipitate in your samples is worrisome. You shouldn't see insolubalities after purification.
http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf
i found this on searching in Internet may this help you:
1.Use Sodium acetate (0.3M final conc, pH 5.2) for routine DNA precipitations.
2. Use Sodium chloride (0,2M final conc) for DNA samples containing SDS since NaCl keeps SDS soluble in 70% ethanol so it won’t precipitate with the DNA.
3.The role of temperature in ethanol precipitation…
Incubation of the nucleic acid/salt/ethanol mixture at low temperatures (e.g. -20 or -80C) is commonly cited in protocols as necessary in protocols. However, according to Maniatis et al(Molecular Cloning, A Laboratory Manual 2nd Edition… 2nd edition?? – I need to get a newer version!), this is not required, as nucleic acids at concentrations as low as 20ng/mL will precipitate at 0-4C so incubation for 15-30 minutes on ice is sufficient.
All the best
After DNA extraction and precipitation, I have done a second precipitation with NaOAc and EtOH after dissolving the contaminated DNA. Result: same crystalline precipitates as in the first step.
The problem occurs since I use NaOAc instead ammonium acetate.
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