pcr contamination is often caused by transfer of post pcr product from the electrophoresis buffer back into your working area on gloves/hands and can get onto dust and surfaces so a clean up of the working area with bleach including all bottle surfaces and shelves in the area. Checker gels should never be run close to the pcr setup area and nothing should come from the post pcr gel area back into the setup area eg leave your gloves in the gel area if you have handled gels. Try setting up a run in another laboratory with your reagents but their equipment to see if it is in your reagent or in the environment. You can get rid of contamination by using one new primer outside of your current primers then the contamination cannot amplify but you will still need to change your working practice or a new contamination problem will eventually start again with the new primer set

positive main aa raha hai na? negative to dekh lenge.... :) 

bhaisab, above advise looks promising and try another primer set with same reagents and prepare master mix at same used area and at different from this.  Also, use all new agarose, buffer and sterilized tank for electrophoresis and load negative control 4-5 well away from positives. What I think is if another primer set is not yielding any amplification in negative, then your specific primers may have very common annealing site.

Keep trying...we will sort it out. 

Dear Sudhanshu,

Above all suggestions are to be followed for PCR. I would like to add just another one. I assume that you are using universal bacterial primers for microbiota studies. Since commercial Taq Polymerases are recombinant proteins, expressed and purified from bacteria, small amount of the host genome may remain in the final Taq preps, which gve you a positive amplicon with universal bacterial primers. I dont know which Taq are you using, but there are highly purified Taqs for similar microbiota projects. you can try those. Else there is a very easy technique, which you can follow. I am attaching the paper link.

Best of luck,

 SD

http://jcm.asm.org/content/37/10/3402.full.pdf+html

@ Paul Ritland @ Vipin Sharma @Anja Hühnlein

Thank you for your suggestions,,, I have done all possible efforts to the best of my knowledge,,,I have changed the primers 2 times, I means I made a new stock of primers and I always run negative after 2-3 blank wells in agarose gel run,,,,I have changed and taken new PCR Buffer, water.....yes it happens when we use plates for PCR ( means evaporation can cause NTV contamination) but for this reason I am doing my experiments in a very controlled way so I am using just PCR tubes to avoid such contamination,,,,I have asked my colleagues to check if I am doing some mistakes, but they are also getting NTV contamination.....I have already set up an experiment in which at each step I am changing one PCR reagent,,,,,,I am attaching the gel picture here. In this, near the ladder is my negative control and you can see that this a strong amplification band. It looks like DNA contamination... but how I am not able to make out after doing all possible efforts....

Hello sir,

I doubt contamination in the master mix being used. Have you tried PCR master mix from other sources? Can I know what primers you are using?  

you can try one thing to figure out the problem. Treat the PCR master mix with DNase. Treat the master mix directly with DNase (10IU/2ul PCR master mix for fina 20ul PCR reaction) at 37 degrees for 30 min, followed by DNae inactivation at 90 degrees for about 30-40 minute. Post DNAase inactivation add the template, primers and water to check the PCR efficiency.

Hope this will help.

Best 

Hii, 

One more thing that you can do, take your DNA, primers and Master mix and perform all your experiment including gel run in your friends lab or any other lab or a lab away from your lab. You should try to avoid Max. things from your lab. If you can borrow water and master mix from other lab, this is also better.  and also, once you should only amplify negative control, nothing else. I hope this can work or could sort out main reason for your problem.

Best wishes

Sorry for the delayed response,,, Finally I got rid of negative amplification ,,, I followed the general practices ,,,did some gradual change of reagents (new reagent at every change and keep the old one ) and one final reaction with all new reagents....I was already using DNase - RNase free water (but for this experiment I ordered new lot of water) And I performed all experiments in a different lab where no wet lab experiments are carried out....I cleaned the PCR hood 2 times with 10-12% Bleach then one time with a solution known remove RNA, DNA, Bacterial contamination and finally with 70% Alcohol and wiped it and dried the surface). Then performed all experiments and did not get the negative amplification.... Thank you all for your suggestions