This is my second Gibson Assembly mix. The third well is the Nebuilder Positive Control and the fifth well is my Gibson Assembly mix. I have done tw fragments of 2.7 kb and the vector which is 3.3 kb. The incubation time was 1h .30 min but still not able to see any fragment around 8.0kb. Also I dont know exactly how should it be seen (size) the positive control assemblied or not, and have not done transformation yet. Please any suggestions is welcome.
You should not be pre-running the assembly mix. It really does not tell you that much since the concentration of the assembled product might not be high enough to visualize but is high enough transform. I suggest you just run the transformation then do colony PCR. If you really want a confirmation before using competent cells you can just do a PCR on the assembly mix the extends over the assembly junctions.
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