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I'm really happy to help anyone troubleshoot their project if it isn't working as expected, but there are a few things you can do to make the process much easier.
1) Include a project ID. The RF-Cloning site assigns unique IDs to each new project, which can be found in the URL (e.g., http://www.rf-cloning.org/rf_cloning_project.php?proj_id=bcb296de55989ff65abb4aad6894d13f)
2) Let me know about your cycling conditions. What polymerase? Any additives? 2- or 3-step protocol?
3) Include, at minimum, the following two controls:
a) A pseudo-positive control, where you transform with secondary PCR product that has NOT been treated with DpnI (we expect a lawn here, or close to)
b) A negative control where no mega-primer is added to the PCR reaction (we expect no colonies)
Happy cloning!
-Steve
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