Anything and everything cloning: Go...
New accounts disabled to block spam. The forum is just an archive now.
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
Offline