I extract DNA from the sediment sample, but the ratio of 260/230 is much lower than 1.7. I use the Nanodrop to measure the ratio. I use some methods such as cold ethanol washing or phenol washing, but the ratio is also lower than 1.7. Can you give me some suggestions to improve my methods. Thank you!
Hi Heaven, could you shortly summarize the whole isolation process?
Phenol has an absorbance close to 230 nm. Hence, the 260/230 ratio might be influenced by phenol still present in your sample.
I'm so sorry that i didn't write down my protocol.
1. i use the Tris buffer add to the microtube that contain my sediment sample and some glass beads;
2. then bead beading the sample; add proteinase K to the tube and keep it in the 37 degree for 30min;
3. then add SDS and keep it in the 60 degree for 2 hours, centrifugate the tube and transfer the supernatant to a clean tube, and then choloform, phenol and 2-Isopropanol were add to it.
4. Centrifugate the tube and transfer the supernatant to a clean tube carefully, use the pH=4.2 sodium acetate to precipate the DNA;
5. Centrifugate the tube and abandon the supernatant and use the 70% cold ethanol to wash the DNA twice, centrifugate the tube and dry the precipitation;
6. the last step is add ddwater to resolve DNA.
in the step 4, i transfer the supernatant to the tube and avoid to touch the mixture of phenol, choloform and isopropanol. so the phenol is not in the DNA which I get finally.
I think the sediment sample have much humid acid and other materials which can absorb the light at 230nm.
I hope to know how to remove these material?
Dear Heaven,
your protocol is in general quite similar to protocols we use. You could easily change your buffer against a buffer containing CTAB (see the cited paper for a suggestion). CTAB is applied to reduce the concentration of humic acids. We do not use phenol for DNA extraction, but a 24:1 mixture of chloroform:isoamyl alcohol. After the extraction we add isopropanol and sodium acetate (pH 5.2) to precipitate the DNA. DNA-pellets still are often brownish and we use a column-based method to further purify the DNA. But that also depends on what you need the DNA for...
Article DNA recovery from Soils of Diverse Composition
Thank you Felix Eikmeyer! We use the tris buffer which cotains CTAB. As you said that the DNA-pellets still are brownish. I did not use the column-based method to clean the DNA, but that sounds good. which column do you use? Do these column bind the big fragment of DNA. (e.g. >23Kb).
we want to get high quality DNA to 454sequencing.
And thank you again for your sended article. Zhou's method is very popular.
Dear Heaven,
we are using the Macherey & Nagel AXG 20 columns together with parts of the Buffer Set III followed by another round of isopropanol precipitation. We also prepared DNA for 454 sequencing this way. But there might be further kits & columns that are also suited.
http://www.mn-net.com/tabid/10743/default.aspx
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