I am currently studying molecular cloning methods and working on understanding the selection process in bacterial transformation. One common challenge is the occurrence of false-positive colonies, which makes screening more time-consuming and less efficient.
Could anyone recommend specific cloning vectors that help reduce false positives and improve reliability during selection? Practical suggestions or references to widely used systems would be very helpful.
Dear Siddhartha Lanke
the false positive are not related to the vector itself but to the cloning method, since each cloning approach may result in a small fraction of undigested or re-ligated empty plasmid (using ligase-restriction enzimes) or template empty vector used for PCR amplification in PIPE cloning for example, that may lead to growth of colonies those are negative to PCR screening since contains the empty vector
In generally the use of an empty vector carring the ccdB gene (in Gateway or PIPE cloning) help to drastically reduce this background, but this could not be appliable to the standard cloning approach using restriction enzime since the ccdb is big and cannot be integrated inside a multi cloning site.
good luck
Manuele
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