I am looking for microbial diversity pattern in soil samples from different altitudes through DGGE. I used FastDNA™ SPIN Kit for Soil DNA extraction but I am getting lot of brownish color in the final eluted DNA. Can anyone suggest me a method to get rid of this problem, as this is hampering further downstream process.
Hey Suvigya
Strong brownish coloration could indicate high amounts of proteins, polysaccharides or RNA contaminants or all together.
You can try to get rid of the proteins, by incubation with Protinase K or try to get rid of all the purities using ethanol based precipitation of DNA. You can also try to add PVPP.
The other option is to try with specific purification kits, which is a bit costlier method.
I hope this can provide some help.
All the best,
D
I did tried with Mo Bio soil DNA extraction kit ...but the color problem remains the same..Thanks for the suggestions !
Did you try to amplify it? What are your DNA concentrations and what are your 260/280 ratios? Maby you can also do a simple dilution series and see will it work then. Of course, it really depends on what you need the DNA for.
possibly the colour is caused by humins which often purify with dna. You could try ethanol precipitation ( with glycogen added if you do not have very much dna as a co precipitant then do an OD260/280 to ascertain dna purity Dusan makes a good point that when you do have interfering substances sometimes using less samples allows a PCR to work rather than using more and having more interference. Having said that any substance that absorbs at 260 will make you think that you have more dna than you actuallu do have
Agree with Dusan and Paul.
I advice you to re-purify your DNA extracts with a good quality Plant DNA extraction kit, starting from the incubation in the lysis solution step. Plant DNA extraction kits are good at removing those co-purifying substances. you may modify the protocol by repeating the first additional 2 times. This can significantly reduce the enzymatic inhibitors. Lastly, if you have good yield, use 10x diluted DNA, instead of the original extract and addition of BSA to the PCR mix may also help you to reduce the effects of inhibitors to a great extent.
best wishes
Thanks Dusan , Paul and Sibnarayan for the inputs. I will try and maybe come back here again !!
Unless you have definitively ruled out the MoBio Powersoil kit as not working for your samples you may want to give it another shot. I have had good success with those kits for a range of sedment/soil samples, often containing LOTS of humic substances and even raw bitumen. but as others have said easiest thing to do initially would be ethanol prec. or even repeated washes to clean elution.
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