I am doing nucleic acid extraction (manual silica magnetic bead-based) from the serum sample. After the extraction, I have taken the OD 260/280 in UV spec. There is no protein contamination. But the purity of DNA is very low (OD 260: 0.014). So, I tried the two different wash buffers to remove the salt in that solution {Wash Buffer 1 (25 mM Tris-HCl, 1.8 M GuHCl, 75% EtOH, pH 6.6 and Wash Buffer 2 (10 mM Tris-HCl, 100 mM NaCl, 80% EtOH, pH 6.6}. Finally washed with 70% ethanol. But I cannot able to get the purified DNA. Please, anyone, suggest to me a good wash buffer for salt removal and purified DNA.
For salt removal, the ethanol wash can be repeated on the eluted DNA. Ethanol precipitation is a good method for desalting and concentrating DNA. Ethanol changes the DNA structure so that the DNA molecules aggregate and precipitate out of solution (see Eickbush and Moudrianakis, 1978). Most salts and small organic molecules are soluble in 70% ethanol, leaving the precipitated DNA ready for separation by centrifugation.
Is 0.014 the 260/280 value ? If so, then it could mean that there is
1. residual guanidine from the kit
2. carbohydrate carryover ( usually from the Glycogen used for preciptation)
3. residual phenol from nucleic acid extraction
In which case you might need to clean up the elution by Phenol chloroform extraction of the DNA and preciptate with isopropanol/ethanol and wash with 70% ethanol.
Dear Praveesh Valissery
Thank you so much for your kind reply....
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