yes. primers could be the reason. formation of primer dimers and secondary structures in primer may also result in bands in the gel. also check your DNA concentration to be optimal before you add them.

Hi Elizabeth,

Some questions from my side, before I can give an advice: What do you mean with "Except that I did the same PCR reactions with different DNA a few weeks ago, and I had good amplification"? Have you amplified this region successfully? Can you please post a picture of the gel?

Best

Ijad

Hi,

if your multiple, aspecific bands are longer then your specific one, a feasible solution is to decrease extension time. You can decrease primer concentrations as well. There are numerous things that you can do to optimize your reaction. Have you tried touchdown PCR?

Regards, Tom

Hi,

you write you have tested different temperatures etc. So I don't think this you don't have to test again. Especially because the PCR works some weeks ago. I think you should first test the DNA from some weeks ago - if you have some of this DNA. If you have here also three bands now, I think you mix different primers and you have to order new ones.

Hi,

First I would like to understand,what is the difference in the DNA in both the reactions.In one it is working well and in second it is giving three bands with the same condition.As you r talking about 16S bacterial gene,people use universal primers to amplify all bacteria for metagenomic studies.

Where are these additional bands? Above or below the correct product?

Is there correct band?

Did you check DNA for degradation? Did you take into account contamination?

Your question has the answer. Untill unless you find what is the difference between the two templates used, the discussion proceeds nowhere. For sure, if no change in rest of reaction composition or the PCR program was done, primer are not the culprit. Just focus on templates for now.

Good Luck and do reply how did you resolve the problem. It may help others.

yup u should change annealing temp and also recheck primer secondary structure formation. I believe you will get your solution

Hello

you are getting clean negative control means no primer dimer then your primer is ok but when you are changing DNA then three bands are coming, so problem is with your DNA.

when you run a PCR product in gel, run your only template in one well. Check and compare your PCR product and DNA template.

West wishes,

Asit Jain

Dear Elizabeth,

You have not mentioned the size of your band and what are primer binding sites in the ribosomal gene. are you getting non-specific amplification along with the desired band or all the non-specific bands?

May be your primers will have more binding sites.

may be one of the primer have more binding sites and you are getting only single strand amplification.

May be you are getting primer-dimers that you could avoid with change in temperature or some additives like DMSO and BSA.

you have to look all the possibilities.

I hope you will get the solution.

I don't really know what happened that time, but I have adjusted the magnesium, and temperature, and checked my DNA concentrations. And I think I fixed it. I am not sure what I fixed, but it hasn't happened again. Thanks for all your advice.

and what's the Ta now on which are you getting "SPECIFIC only " amplification.

Thats really good..........any how u got solution

Sorry it took me so long to get back. I started testing my PCR using a different sample, which worked, so I suspect something happened to my original test sample. I now use 27f and 519r 16s rRNA gene primers, using 56C for my annealing temp. I am using 5ul of 25mM MgCl2 in a 50ul reaction. I also use 25 cycles, instead of the 30 I was using before.

I hope that helps.