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Natalia Oliveira 09-07-2007 06:44 AM

Artifactual double bands
 
I would like to know if anyone has any information on "artifactual double bands"? The only paper I could find that refers specifically to this event, talks about it being observed in DGGE analysis of PCR products that did not have the final elongation step 72C extended (Ingmar J. et all).
Now I'm working with a derivative of pGEM plasmid to which PCR rxn was run at 3 different temperatures (54C, 52C, 50C), and a regular 0.8% agarose gel was run at 104 Volts, which resulted in bands very much like "artifactual bands" (primers were designed and highly specific to insert). Could that be possible? Would the artifactual double bands be an artifact of the DGGE analysis? Does anyone have another suggestion for the observation of double bands on PCR rxn? Also I would like to point out that the band was very but very slightly bigger than the amplified fragment, and it was more distinct on the gel as the temperature decreased (more at 50C than 54), which was also tru for the fragment amplified ( way more intense than the supposed artifactual double band).
Thanks


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chovek69 09-08-2007 11:28 AM

Artifactual double bands
 
On 7 , 09:44, Natalia Oliveira <[Only registered and activated users can see links. Click Here To Register...]> wrote:

Hi,
I also have troubles with artifactual double-bands. (see this thread
[Only registered and activated users can see links. Click Here To Register...]).
However since one of my primers is Cy5-labelled I was thinking that
the problem is in the different MW of the two chains of the PCR
fragment. Now that I've read the paper of Ingmar J. et al. I assume
there is something more. In my case the artifactual bands are 3-5bp
smaller than the real band and if the Cy5 is the problem why the bands
are of different intensity. In your case it is obvious that the
secondary structures are the problem as when you decrease the temp
they become more prominent.
The authors give interesting ideas and I will test their solution. Did
you test it yourself ?



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