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).
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?