May be you could dilute your extracted DNA (to reduce PCR inhibitors such as humic acid) and than use several microliter as a template to have around total 10 to 30 ng DNA per reaction. Usually 10 t0 30 ng DNA per reaction should be more than enough to get the 16S rRNA gene amplification. You could also setup some PCR reactions with the extracted soil DNA (as you are doing now) and add pure culture genomic DNA (that you are using as the positive control) and see whether you could have amplification of 16 S rRNA gene of the purculture DNA or not from this mixed reaction. This is a type of spiking study to determine the presence of PCR inhibitors in the soil DNA. By the way what kind of soil was that? Did you use Nanodrop to quantify DNA, if so, what was the 260/280 or 260/230 ration. Good luck. Tanvir

The concentration what you have obtained is low. How does the DNA look on the gel? I suggest you to do an ethanol precipitation and concentrate the sample before you proceed on to the 16S amplification.

You may be unlucky enough to come across a bacterial population with rich GC content in their genomes. Have you tried adding DMSO (3%-5%) to your PCR?

The most important consideration is already commeted by MD Tanvir Rahman. it is very important to see the quality of DNA, interference by humidic acid...and salt concentration in your DNA sample. In the market you can pass though your DNA by desalinization column. or just wash your DNA precipiting with alcohol or isopropanol,

washing with 70% ethanol.....

run bioanalyser gel to check quantity and quality,

by PCR you can have results from 5 ng if you use a platinum or golt taq polymerase, before rum pcr make heat shock (at 70C) in 10 minutes before pipete sample. add if possible 1% DMSO, and increase a liltle bit of magnesium or manganese in the buffer (depend the taq you are using)

try in longer cicler and change the delay time of your termoclycler program

good luck

mario

As Lekha suggested your DNA concentration seems very low... you use 100-200 microlitre of your sample and precipitate it with ethanol. it will reduce impurities as well as concentrate the DNA. then again check the quality of the DNA and use positive control for PCR reaction.

Hope after this you will have the results positive. Because I was also had same problem and finally solved.

Thanks for the valuable inputs everyone. Dr.Md.Tanvir Rahman I did carry out the reaction by mixing together my metagenomic DNA (5μl) + pure culture DNA (5μl) = 10μl mixed DNA. And then carried out PCR with this sample by taking 3 μl as template DNA in three replicates ( total 9μl ). Along side I did take my pure culture DNA as positive control in another reaction tube as always. The results showed no amplification at all in the 3 replicates using mixed DNA but positive control DNA again showed good clear thick amplification. The experiment suggests presence of inhibitors in metagenomic DNA or may be my pure culture DNA got diluted in the mixture.

Next I intend to purify my DNA using High molecular weight DNA purification kit from Zymo research as suggested by Kamlesh Jangid (NCCS, pune)

Have you tried using CTAB method, in some cases where the kit based extraction have gone bad, the non-kit based technique worked better (It was both in forest soils and agricultural soils). We have used MoBIO power soil kit.

Using of BSA (1 μl) in a 50 μl reaction would make your PCR work, but sometimes without BSA would be better, provided the sample is diluted. Moreover the amount of DNA you have measured is from the soil which could be from plant roots, insects, insect eggs or even nematodes, fungi and bacteria . With the kits, sometimes the rare taxa are lost, but the quality of nucleic acids would be cleaner. When you use the traditional method (CTAB) the quality of nucleic acids wouldn't be cleaner but you can get the rare taxa amplified.

Try using Molbio FailSafe PCR kit. It has several combinations of inhibitors and enzymes. We have used it in our lab and it has been successful.

Rajal Debnath,

How did you know your environmental DNA prep being pure? That is, what did you do to arrive at the conclusion that your DNA prep is pure.

Suggestions:

1. do a series of template dilution amplification: eg. (1) original (the way you have been doing so far), (2) 1:10 diluted template, (3) 1:100 diluted template, (4) 1:10000 diluted template, (5) 1:100000 diluted template, and (6) 1:1000000 diluted template. Remember, keeping all other components same and their concentrations the same.

2. Describe your gel pictures when you get results from this series template dilution tests, as well as your previous tests if you can. When taking the gel picture, try to get a series of longer, and even longer exposure to get a clearer view of the gel. A "right" exposure does not help you troubleshooting.

Hello everyone. Finally got amplification. As I was waiting for my Zymo kit to purify my metagenomic DNA. I thought of isolating the DNA again using MoBio Powersoil kit. But this time in an attempt to increase the amount of DNA, I processed three different samples and passed them through the same spin coloumn. The elution resulted in DNA concentration of 52.9 ng/μl, 64.9 ng/μl, 45.6 ng/μl ( measured by Biospectrometer, Eppendorf) . All the three samples were used for amplification giving a positive signal. My previous samples which gave me negative results also had sufficient amount of DNA when i measured with the same instrument. but it was due to the humic acid and other inhibitors that resulted in negative signal. Thanks a lot. and am on my way to cloning the fragments!!!!

congratulation Rajal

Thanks Mr. Nageena. Your feedbacks were really helpful.

Just a suggestion for future DNA extraction using MOBIO power soil kit , use a genogrinder instead of a vortex and if possible more than one extraction from the same sample gives a greater concentration of DNA to begin with, a colleague of mine Dr. Feinstein and a Prof in our dept Dr. Chris Blackwood has published this information in a paper (Feinstein, L.M., Sul, W.J., Blackwood, C.B., 2009. Assessment of bias associated with incomplete extraction of microbial DNA from soil. Applied and Environmental Microbiology 75(16), 5428-5433) .....All the best !!

You can try your reaction with DMSO, BSA combinations and same time dilute your DNA at different concentrations and try. Because humic acid will inhibit the rxn, if i dilute the DNA there may be chances to amplify it.

I am also facing the same problem, Used Favorgen Kit for Soil DNA extraction but it was not effective then I used manual method for DNA extraction which was effective but unfortunately due to inhibitor did not get PCR results, I run 200ul DNA on 0.7% agarose gel mixed with 2% PVP and then purified the DNA By Promega kit and check the pcr with different dilution ...... but still out of track .....work is pending

Any suggestion/commends will be appropriate !

Thanks

Naseer

Follow my suggestion above, then we can discuss the problems you are faced with.

Generally Soil DNA contains a number of PCR inhibitors. We have stried sometime DMSO and try to keep the concentration of your DNA less in PCR. you can try 0.25 microliter of sample.

I would first try diluting your DNA, say 1:10, 1:20, 1:50, and 1:100, and see if that helps. As suggested above, diluting might help in case of inhibition. To see if inhibition is the culprit, you could also make a dilution series of a positive control, and amplify that by itself, and in the presence of 1 ul of your DNA. If the positive bands of the series end at a much higher concentration in the presence your your DNA, you have inhibition.

Another cause might be breakage of DNA during extraction. In that case, you might switch to a shorter PCR, say 8F x 515R or 8F x 806R. These shorter PCRs are usually more sensitive and will give better results in difficult situations, but they will still give you a good stretch of sequence for phylogenetic analysis.

Good luck!

Rajal i am also facing the same problem. Did you also purify your DNA before amplification

Try the new PureLink Microbiome DNA Purification Kit – it has robust protocols for soil etc http://www.thermofisher.com/order/catalog/product/A29790?ICID=search-product