I am using FastDNA SPIN kit for soil to extract DNA from sludge samples. However, the A260/A230 ratio is very low (i.e. 0.2), which signifies contamination of humic substance. Does anyone use the kit and experience the same problem? How did you rectify the problem? I am afraid that this low ratio would hamper the quality of my downstream processes using this extracted DNA.
Please let me know the samples details and the initial steps you are taking for removal of waste
The samples are raw sludge taken from local wastewater treatment plant. During samples preparation, the sludge was washed several times and resuspended in TE buffer. DNA extraction was conducted according to the protocol as outlined by the kit supplier.
This is a typical result when DNA is extracted with FastDNA Spin kit for soil and the yield of DNA is low. This is caused by something unknown which is bound to the DNA and often such DNA-extracts have PCR-inhibiting factors. This can be overcome by diluting the DNA-extract when used in PCR-reactions.
Thanks Frida. The strange thing is the DNA concentration is quite high as determined using nanodrop. You have mentioned one of the solution for that, which is to dilute the DNA-extract when use in PCR. ^^
There are a few strategies you can use for equating the presence of humic substances and other potential PCR inhibitors. Sample dilution is one fast choice, but, when this does not produce a good result, you may try running the total DNA extract on agarose. This will separate humic substances from DNA and you will recover DNA from the gel by cutting the band, melting the agarose and precipitate DNA with ice cold ethanol (however, there are kits for that too).
U CAN TRY NUCLOSPIN SOIL DNA EXTRACTION KIT..It works well for extracting high quality DNA from sludge
http://www.mnet.com/Portals/8/attachments/Redakteure_Bio/Protocols/Genomic%20DNA/UM_gDNASoil.pdf
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