Hi

It is hard to say without looking at the gel and knowing what size are you aiming to and what size you actually got. I find that sometimes when running an amplification product for one of the 16S variable regions student often confuse primer dimers with extra bands. This is especially the case for bar-coded primers. Furthermore in some cases you could get natural size variation since you are looking at a community, this normally results in hazy unclear bands (again hard to say if this is what you mean with out looking at the gel). This could also be the result of non-specific binding of your primers or amplification of you polymerase if not a hot start one.

In any case, my suggestions to you are:

A) In case you are not working with a hot-start polymerase, work on ice all the time, pre-heat the thermocycler block to 94 and only then move the reaction tubes to the thermocycler.

B) Try lowering your DNA concentration or your primer concentration or both, this helps in the case of non-specific binding of primers.

C) Try doing a touchdown PCR, by that I mean that in the first 10 cycles the annealing  temperature drops by 1 deg C per cycle, starting at 65 in cycle one and reaching to 55 deg C by cycle 10, after that you continue for another 20 cycles at 55 deg C. This allows for a more stringent conditions at the early stages of your PCR reaction where the template is mainly genomic DNA and is likely to cause more non-specific binding of the primers.

Hope this helps

Ido 

Hii...

The multiple bands with 16S rDNA primers is only because of mis-annealing of the primers due to low annealing temperature. So try gradient PCR upto 65 degrees. you should get the clean product from 55-60 degrees.

Hope this should work..

Best wishes..

Since this is metagenomic sample, is it possible that some bacteria have shorter 16S region (for example shorter v3 region)? That's why this resulted in shorter band?

which primers did you use? I have problems with 515-806 primers, so I cut the band I need in the last step. but I still think there should be some improvements could be done to optimize the PCR.

The best results for separating 16s rRNA in bulk samples are obtained by doing a touchdown PCR with GC clamps on the forward primers. This would result in good separation of bands from each other. Try denaturing gradients of 30-70% in DGGE gel for optimal resolution of bands. 40-60% is also advisable in case too much of PCR product is there. Try and limit the concentration of DNA in template to about 70ng/uL. Anything more than that would result in smudgy bands due to over amplification of template strands

Well. There are a number of microbes with several copies of the 16S rRNA gene, and differences between these copies can sometimes be quite significant. For example, in some haloarchaea these copies can vary by a few per cent (e.g. Cui et al. 2009 Extremophiles 13:31-37). But even if the differences between the copies are very small they can potentially form different bands on a DGGE gel. An example are Alteromonas isolates from the Mediterranean (Sass et al. 2001 Appl Environ Microbiol 67:5392-5402). When the first isolate produced seven bands that was explained by it not being a pure culture. But when you have a range of isolates with the same bands it is becoming suspicious. Seven times the same organisms with the same 6 contaminants?! The bands were excised and sequenced and it became clear that they were very similar (>99.8%), in some cases only 1 base different. So, you can try to get your conditions more stringent. But to be really sure what you have you need to sequenced those bands. Good Luck.

Note that you guys are talking about different bands on a DGGE with separates pcr products based on %GC and to lesser extent sequence, where as the question is bands of different size coming out of a PCR reaction.   Yen, it sounds like your problem is PCR optimization. though there might be size variation you shouldn't get multiple bands. However as I mentioned earlier, for you to get the best answer you need to attach an image of your agarose gel with as many details of protocol as you have. 

Did you run a negative and positive control, its easier to troubleshoot that way.

There are a many things that can go wrong.

In case you see multiple bands in negative and positive controls...yes you have a problem, else its just contamination of your reagents.

Dear all,

Thank you for your suggestion. I have found out the reason. Since my sample is metagenomic sample with a mixture of DNA, it is normal to have multiple bands as the length of 16S region will be different for some bacteria. I constantly get two bands (1500bp and 1400bp) for this sample.

Do you have documented evidence of such a large size variation on the 16S sequence ? I find it quite strange and I've never seen such a huge size difference on full length 16S PCR from soil samples.

Additional bands may reveal the presence of amplified eucaryotic DNA, since some 16S primers were shown to bind for instance to spots in mouse genome. I would try to purify the additional band from the gel, sequence it, and BLAST to securely determine the source. 

Any updates on your experiment and the multiple bands? i am having the same problem right now...

Hi, I have had the same problems. I did a gel extraction and selected the larger band (maybe the smaller band is not fully amplified?). However, I am not sure if this is correct. If I have barcoded PCR products for Illumina sequencing, can I send a pooled sample that has both bands? I am using F515 and R805 primers for 16S, but my amplicons are either 425bp or 525bp... which band should I chose?

Last time we just sent the pooled samples with both bands. According to one of the senior student in our lab, he said it is normal to have multiple bands in metagenomic samples since the size of 16S region can be varied.

I also had just 1 band but now they are 3 when using the 515F and 926R primers for Illumina Miseq

I also get 3 bands for the 515F and 926R primers for Illumina Miseq

I got two bands only for a specific sample. my primer is 27F and 338R. But in the other sample it's perfectly fine. What is the possible reason for this?

May I ask what primer have you used?

Recently I'm using 515-806 primers with no adapters but got 2 bands, both at 200~300bp.

Is that normal to have multiple bands using 515-806?