Hello, I`m working on a proyect that requires a high purity DNA for sequenciation. Right now I`m trying a soil DNA extraction with NTES buffer, phenol-chloroform-isoamilic DNA and doing the entire process at 4°C and I`m getting ratios of 260/280 and 260/230 of 1.6-1.3 when checking the DNA with a Nanodrop equipment and Ieed to have a concentration higher than 100ng/ul with a ratio of 1.8-2.0. I`ve tried ethanol 96% washings and isopropyl but i end up with the same ratios and lose a lot of DNA.
So I`m looking for any tips or help to make my DNA more pure without losing a lot.
Thanks.
I think that you are not losing DNA. There are many complex organic compounds like humins in soil which both look like and purify like DNA and you are getting these compounds purifying with your dna which accounts for the poor OD ratios. Having partly purified a lot of material it may be necessary to complete the purification with one of the many column purification methods on the market. I attach some general information about soil DNA which may be of interest
Well, for some reason when doing the process at room temp the interface does not form. And at 4C i'm able to see it and take the sobrenadant before it disappear.
Often, I extract DNA from soil with Qiagen kit and do get pure DNA (purity of 1.8 @ A260/280 with nanodrop) and very good yield except one occasion (light brown DNA sample) which i ran through column of high pure PCR product purification kit and it was fine, alternatively you may use a clean up kit though may loss some DNA so it better if your DNA concentration if very high.
Also, you may retry the ethanol precipitation using LPA and washing twice with 70% molecular grade ETOH.
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