Difference between revisions of "Problems with PCR using VR/VF2"
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==VF2 Incorrect Annealing== | ==VF2 Incorrect Annealing== | ||
| − | [[Image:VF2annealing.png|thumb|left|]] | + | [[Image:VF2annealing.png|thumb|left|Diagram of potential VF2 binding sites within R0062]] |
| − | VF2 can bind | + | VF2 can potentially bind [[Part:BBa_R0062|R0062]] in two different places. Annealing to the segment shown in blue in the diagram seems unlikely to result in extension since there is not any significant pairing near the 3' end of the primer. Depending on the annealing and extension temperatures being used, VF2 may prime from the segment of R0062 shown in red. The binding would not be strong, since the matched bases are mostly A's and T's, but it could be enough to produce the excess bands in the PCR. |
| + | [[Image:VF2 1.png]] | ||
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[[Image:VR 2.png|thumb]] | [[Image:VR 2.png|thumb]] | ||
[[Image:VR 3.png|thumb]] | [[Image:VR 3.png|thumb]] | ||
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[[Image:VF2 2.png|thumb]] | [[Image:VF2 2.png|thumb]] | ||
Revision as of 16:39, 21 August 2007
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
VR can potentially bind B0010 in three different ways. One of the annealing schemes seems particularly likely. Only 3 of the last 13 bases of VR mismatch to B0010. Also, VR's 3' end would anneal the the part tightly due to the two G - C pairs.
VF2 Incorrect Annealing
VF2 can potentially bind R0062 in two different places. Annealing to the segment shown in blue in the diagram seems unlikely to result in extension since there is not any significant pairing near the 3' end of the primer. Depending on the annealing and extension temperatures being used, VF2 may prime from the segment of R0062 shown in red. The binding would not be strong, since the matched bases are mostly A's and T's, but it could be enough to produce the excess bands in the PCR.
