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I want to run multiplex PCRs for my genotyping, with a primer pair targeting my construct and a primer pair targeting some housekeeping gene (sort of a built-in control).

I designed the control amplicon to be very short (I tested 3 primer pairs with ~120, 85, and ~50bp amplicons respectively). The main rationale behind this being that usually my amplicons are ~200-400bp, and I want something clearly distinguishable, but shorter rather than longer (I want to keep my options open for longer target amplicons).

In any case, regardless which control primer pair I use, whenever I try to multiplex I only end up seeing the shorter amplicon. See the gel picture below for an example (lanes: 1-ladder, 2,3-target+control, 4,5-control, 5,6- target).

enter image description here

I see that lanes 2,3 have a very faint additional smear compared to 4,5, but I want a band for my target amplicon. Also, my shorter bands are quite blurred.

Also, just for reference, my amplicons do not overlap at all.

So, I guess my questions are:

  • Why is the longer band disappearing?
  • Why is the shorter band so blurred?
  • What can I do to prevent this form happening?
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  • $\begingroup$ How far are the construct and the housekeeping gene from each other? If they are close there might be transcription interference occuring. $\endgroup$ – Nandor Poka May 4 '15 at 11:50
  • $\begingroup$ As long as the amplicons do not overlap, I don't see how there could be any direct interference. In any case, they do not overlap and are on different chromosomes. $\endgroup$ – TheChymera May 4 '15 at 11:52
  • $\begingroup$ Overlap is not necessary for transcription interference to occur, but since your genes are on different chromosomes it should not be the case. Did this setup worked before or this is the first use of these set of primers? $\endgroup$ – Nandor Poka May 4 '15 at 11:58
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    $\begingroup$ What are the $T_m$ of these primer pairs? If one primer pair is significantly more stable at a given temperature, it could explain your results. $\endgroup$ – March Ho May 4 '15 at 12:36
  • $\begingroup$ 58-60°C for all primers. All 3 PCRs are run under the same conditions - would you say that there is no polymerase left to be recruited by the less stable primers? $\endgroup$ – TheChymera May 4 '15 at 12:40
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It turns out that the $T_m$ was in fact the issue, or rather, that the computation algorithm overestimated the $T_m$ of the primer pair for the longer amplicon (or underestimated that of the shorter one). At any rate, it seems that at 60°C the shorter amplicon primers hijack the polymerization mechanism.

Setting the annealing phase temperature to 55°C solved my issue, and now I consistently get better results:

enter image description here

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