Smearing on Gel - PCR product shows up as smears on gel (Sep/03/2004 )

Thank you for your replies. It turns out the carry-over contamination may infact have been the problem. After making up fresh solutions and ordering new primers, taq, dNTPS's etc, using a new set of pipets, and setting up post-PCR in a different lab (don't know which of these or how many of these solved the problem) the streaking has disappeared and the bands are bright.
The Mg2+ concentration I'm using is 1.6mM which I haven't changed so I don't think that affected it but thank you for the suggestion, and the thank you for the troubleshooting site recommendations Ihab. Very helpful.



Kudna

Hi Kudna,

it seems you solved the problem, however just an additional comment:

Occationally you can run into PCR primer/amplicon designs where the amplicon can in some way prime itself, to generate concatanated products which will accumulate in number and size as the PCR cycles, hence the smear. This can depend on the exact specificity of teh reaction and hence seem to reappear quite ramdomly, especially if the priming is just borderline.

What are your sequences? Try to check if there is an internal repeat, which would allow the PCR product to act as a primer on itself.

Søren

Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
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www.ProbeLibrary.com
One Real-time PCR kit, which covers 38.565 genes

I think "smesme" has given some theory behind smearing, which makes sense. I like it.

Hi there,

I think i may be having a similar problem - my pcr has randomly started appearing as smears. If this was due to an internal repeat in the amplicon is there anything you could do to prevent it?

Thanks

M

Dear Maza...

I think, you should redesign your primer. It should specific that amplify the target gene only. otherway, it will produce primer dimer. However, before do taht, try to increase the annealing Tm.

the PCR trouble shooting link sent to kudna by ihab is great
thanks ihab
and for kudna im happy u solved ur problem, i had a simillar one and turned out to be that the TAE buffer we are REUSING has exhusted it self
things got better afterpreparing fresh solutions

It seems that this topic has been answered to death, but I had a thought that no one else brought up, and it may help someone in the future?

Oligos will degrade over time, even when properly resuspended in nuclease free water or buffer and aliquoted and stored at -80...eventually they will degrade and this could cause non-specific priming. Based on my personal experiences in the lab, I would consider this problem first if I began to see smeary gels, particularly if I were careful about good technique everywhere, especially if the problem seems to get worse over time.

I had a similar experience once and spent a few months going through every detail of my protocol...remade and reordered my other solutions 8 trillion times and was very frustrated...and it just turned out to be oligo degradation.

hi
do mean by oligonucleotides the primer stock prepared ???
and if that is the case how can u maaake sure that ur primer stock is dead???????