Troubleshoot PCR smear problem - all I got was smear!! (Aug/18/2004 )

yes, of course! I do have the same buffer in the gel as in the gel box.
Thanks.
Kudna

Your problem sounds like non-specific amplification to me, have you tried raising the annealing temperature and/or lowering the primer concentration in your PCR. Another trick would be to lower the DNA concentration in the reaction, sometimes too much DNA can cause these sorts of effects.

I agree with Bob1, but what is the theory behind smearing? Why do we get amplification of fragments of evenly distributed size which show up as a smear? Sometimes you just could not find a clue.

What concentration of Mg2+ are you using? Too much Mg2+ can cause smearing and decrease the specificity of the annealing. The optimal range is 1mM-2.5mM

Hope this is helpful

Ihab

p.s. check out this PCR troubleshooting site
http://info.med.yale.edu/genetics/ward/tavi/PCR.html

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
DK-2950 Vedbaek
Denmark

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