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#1 2016-05-01 14:28:19

Steve Bond
Registered: 2014-01-23
Posts: 129

Troubleshooting assistance: best practice

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!


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