Problems with PCR using VR/VF2

Results of a Colony PCR - the excess bands are shorter and dimmer than the expected product. Columns 3-6 is S03582, columns 7-10 is I13033.

VR and VF2 (Parts G00101 and G00100 respectively) are used to verify the lengths of parts. While using them to PCR potential parts (S03582 and I13033, both composite parts) for the Registry's Assembly Ladder, I noticed that I was consistently getting excess bands. Contamination was ruled out since the bands still appeared in a Colony PCR. Upon inspecting the sequence of the two parts, I found that each contained regions that the primers could bind, resulting in the shorter bands I was consistently seeing. Since S03582 and I13033 are both composite parts, I determined where exactly the primers were binding to see what parts would be affected during PCR with VR and VF2.

VR Incorrect Annealing

Diagram of potential VR binding sites within B0010

VR can bind to B0010 in three different places.

VF2 Incorrect Annealing

VF2 can bind to R0062 in two different places.