Can DNA polimerase amplify primers itself? - need help please!! (Oct/27/2006 )

I am having trouble with my PCR.

My PCR product should be 237bp, although, my water control with polimerase shows a band on about the same size. I made sure the water was ok, it was autoclaved and UV-ed, so no DNA should be there, but the same thing happened. The thing is that without polimerase there was no amplification, so,, what is happening??? is the polimerase amplifying the primers???? can that be possible????


here is the gel, can anybody tell me what is going on and how to fix it please?
I am puzzled....

I am having trouble with my PCR.

My PCR product should be 237bp, although, my water control with polimerase shows a band on about the same size. I made sure the water was ok, it was autoclaved and UV-ed, so no DNA should be there, but the same thing happened. The thing is that without polimerase there was no amplification, so,, what is happening??? is the polimerase amplifying the primers???? can that be possible????


here is the gel, can anybody tell me what is going on and how to fix it please?
I am puzzled....

[attachment=2053:attachment][attachment=2054:attachment]

well i think it's the required size.
Try new batch of bufer and polymerase and a new aliquot of primer.
For me it's a template contamination.
For primers dimers, migrate more. The primer dimers will go fast than 00bp.
Finally if you suspect primer dimer, heat 56° 5' your sample and load it directly.*

Well, I think is in the 200 hundreds, bc the second band from down to top of the marker is 200, and I am looking at the upper band in the PCR reaction, not the band at the botton which is 100. I suppose those are the primer dimers.

QUOTE (medchemgirl @ Oct 27 2006, 05:08 PM)

I am having trouble with my PCR.

My PCR product should be 237bp, although, my water control with polimerase shows a band on about the same size. I made sure the water was ok, it was autoclaved and UV-ed, so no DNA should be there, but the same thing happened. The thing is that without polimerase there was no amplification, so,, what is happening??? is the polimerase amplifying the primers???? can that be possible????


here is the gel, can anybody tell me what is going on and how to fix it please?
I am puzzled....

[attachment=2053:attachment][attachment=2054:attachment]

polymerase doesn't amplify primers.
the band you see is not 237 bp.
maybe you see primers and dimer of primers?
what do you think bioforumers?

You definitely have dimers/concatamers going on. It is clear you are adding way too much primer to your reaction. Try lowering the concentration of primers to 300 nM, you'll likely lose some of the dimerization.

Well, I ruled out template contamination by using new autoclaved and UVed light and still it shows a band in the water sample.

Ok,, I'll try that, thanks.