PCR problems, huge artifacts, no product

Hi,

Been working with this problem for about a month now and haven't managed to solve it. Hoping a first-timer can get some help here.

My primers target the 16S gene and are F: 5'-AGA GTT TGA TCC TGG CTC AG-3'; R: 5'-ACG GCT ACC TTG TTA CGA CTT-3'.

Whenever I run my PCR there is always a product that is under 100bp (proper product: 466bp) which I assume is either a primer dimer or misprime event. I will get a product band most times but there is always this false product band even in my water control. I'll attempt to post a picture.

I have tried adjusting the [Mg++], annealing temp, volume of template, concentration of primers, DNase treated my PCR reagents, and switched to fresh reagents. Nothing has really worked so far. I have checked my primers with the proper software and thought that the primers would be protected against homo and hetero dimers as well as hairpins. This has stumped me and I would appreciate any ideas and help that you guys can offer me. Thank you

riley, how low have you gone with primer concentration? have you tried to titrate a range?

When you say you have a water control, do you mean where you put water instead of DNA and you still have primers there? If so, I would say that is primer dimers. To test this, you could do a reaction without primers: I do this for Real-time PCR and I check the dissociation and the amplification. If you have the possibility to run a real-time, it would be worth to do these controls.

QUOTE (dnafactory @ Sep 13 2006, 04:09 PM)

When you say you have a water control, do you mean where you put water instead of DNA and you still have primers there? If so, I would say that is primer dimers. To test this, you could do a reaction without primers: I do this for Real-time PCR and I check the dissociation and the amplification. If you have the possibility to run a real-time, it would be worth to do these controls.

Yes, it is water and primers with no DNA. I would assume that it is dimers also but the length of the fragment is longer than our primers put together. I don't exactly understand when you say a reaction without primers. Could you explain that more.

I don't have access to RT-PCR but I am developing a technology to mimic rtPCR so hopefully in a few weeks I'll have the prototype up and running, but until then it is only conventional PCR.

QUOTE (riley290 @ Sep 14 2006, 08:11 AM)

QUOTE (dnafactory @ Sep 13 2006, 04:09 PM)

When you say you have a water control, do you mean where you put water instead of DNA and you still have primers there? If so, I would say that is primer dimers. To test this, you could do a reaction without primers: I do this for Real-time PCR and I check the dissociation and the amplification. If you have the possibility to run a real-time, it would be worth to do these controls.

Yes, it is water and primers with no DNA. I would assume that it is dimers also but the length of the fragment is longer than our primers put together. I don't exactly understand when you say a reaction without primers. Could you explain that more.

I don't have access to RT-PCR but I am developing a technology to mimic rtPCR so hopefully in a few weeks I'll have the prototype up and running, but until then it is only conventional PCR.

What he's trying to tell you is to run a "negative control" lane with no primers added to the reaction mixture, i.e - water, buffer, MgCl2, enzyme with no primers present. You will need to make up a seperate mastermix for these negative controls. If no band appears around the 100bp mark, you'll know the problem lies with the primers.

Ok, should I include template in this control or not?

PCR problems, huge artifacts, no product - involves 16S primers (Sep/13/2006 )