16s RNA of metagenomic DNA - soil bacteria (Aug/07/2008 )

I like to do 16s of my soil bacterial metagenomic DNA. My DNA conc is about 140ng/ul.
But I failed to get desired band after pcr!!!!!
85-5',94-2',55-45",72-1' pcr primers were universal primers named 907rU and proRU.

I tried 1/10 dilution of the dna. But no result.
How much DNA is better for pcr????

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try 100x and 1000x DNA dilutions. Primarily, you are diluting the PCR inhibitors, and you have much more DNA than you need for PCR. Do you have these primer sequences? You might want to look at this summary:
http://openwetware.org/wiki/Bacterial_species_identification

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Soil samples have humic acids and other PCR inhibitors. As phage434 suggested, you should try 100-1000 dilutions of the DNA template. If it still doesn't work, I suggest you use AquaGenomic and AquaPrecipi protocol to extract and purify the DNA from your soil samples.

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Are there any effective protocols for DNA extraction from soil which do not use commercial kit? Thank Chessplayer

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I usually use ,< or = 500ng DNA for PCR if the metagenomic DNA is very pure. Is your DNA free from humic acid? Impurities can interfere with your PCR. Is your stock DNA dark in colour because of humic acid? Try more serial dilutions if you think has more contaminations. Otherwise, 140ng/ul is really enough for PCR; even one ul from your stock would be okay.

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Sorry for the delay, and I didn't check the thread. I can send you a sample and if it works for you, we can donate you a kit.

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