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| − | [[Image:070815.png|thumb|right|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.]] | + | #redirect [[Help:Primers/Problems using VF2 and VR to amplify BioBrick parts]] |
| − | VR and VF2 (Parts [[Part:BBa_G00101|G00101]] and [[Part:BBa_G00100|G00100]] respectively) are used to verify the lengths of parts. While using them to PCR potential parts ([[Part:BBa_S03582|S03582]] and [[Part:BBa_I13033|I13033]], both composite parts) for the Registry's [[Assembly Ladders|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. | + | |
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| − | ==VR Incorrect Annealing==
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| − | [[Image:VR annealing.png|thumb|left|Diagram of potential VR binding sites within B0010]]
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| − | VR can potentially bind [[Part:BBa_B0010|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.
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| − | [[Image:VR 1.png]]
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| − | ==VF2 Incorrect Annealing==
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| − | VF2 can bind to [[Part:BBa_R0062|R0062]] in two different places.
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| − | [[Image:VR 2.png|thumb]]
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| − | [[Image:VR 3.png|thumb]]
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| − | [[Image:VF2annealing.png|thumb]]
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| − | [[Image:VF2 1.png|thumb]]
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| − | [[Image:VF2 2.png|thumb]]
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