Have you checked the DNA concentration either by nanodrop or by mere observation in agarose gel. Is there any brown tinge after you have suspended your DNA either in water or buffer, If so you need to dilute your DNA further or purify it by phenol:chloroform extraction for two three times and dilute it further and try doing PCR. you will get amplification.

Do you have used a commercial kit? It's an option ;)

Thanks Baskaran and Rosa. I checked the concentration in agarose gel. there was about 50-60ng/5microL. ya there are borwn tinge when i suspended DNA in water. Once I tried to re-purify it by phenol -chloroform but lost all the DNA. is any other way? please

how you come to a conclusion that you have lost the DNA from the sample. what is the step you followed to re-purify? brown tinge is nothing but the impurities that are interferes your PCR reaction. Dilute the DNA to 10 fold i.e take 10 microlitres from the main stock and suspend it in 90 microlitres of sterile water and do PCR. Even if you didnt get any amplification, what is the protocol you followed for re-purification?

Dear Baskaran, ofclourse after gel electrophoresis i drawn my conclusion. I tried with Phenol (saturate with tris-cl, pH 8)-chloroform (1:1 ratio) and then precipitated with chilled 99% ethanol. but not tried with diluted DNA samples it may work. well thanks for suggestion.

Just use the MoBIo power soil DNA extraction kit (up to 10 grams of sediment) and clean your DNA to remove all PCR inhibitors notably humic acids with the MoBio power clean DNA clean-up kit. You still might have to dilute your sample up to 10 x even after this purification to out-dilute all your PCR inhibitors. Don't measure DNA concentrations with the nano drop especially when you expect low biomass and low DNA yields. Use a fluorometric methods instead (pico green). Much more specific than nano drop.

Thank you Dr. Marco

However, if you decide to use your own method I would use a standard Tris/EDTA/NaCL/SDS extraction buffer and bead beat (zirconium beads) your sediment first. Then incubate with lysozyme (check incubation conditions online) followed by an o/n incubation with proteinase K at 50 degr C. Then extract with PCI 25:24:1 pH8, and precipitate your DNA with 100% ethanol in the presence of NaCl. Wash your pellet with 70% DNA, centrifugate again, dry the pellet and dissolve it in TE pH 8. Your DNA will still be super dirty but you can clean it with the MoBio power clean DNA clean-up kit. Further dilution (~10x or more) of your sample prior to PCR might still be necessary to avoid PCR inhibition. Of course details are missing such as concentrations and incubation times but these are all pretty standard and you can find that info online. The benefits over the use of the MoBio power soil DNA extraction kit (my earlier answer) is that you can play around with sample volume and perhaps improve the extraction efficiency via the enzymatic treatments (which are not part of the MoBio kit). However, in my experience your own method is not per se cheaper than a commercial kit because the costs of ingredients and consumables do add up.

please note typo: "wash pellet with 70% ethanol" of course (not with DNA).