Cloning problem

dygibbs at hotmail.com dygibbs at hotmail.com
Sat Nov 9 21:20:49 EST 2002


I have attempted to clone a 14 kb insert (Mlu I/Xho I) into a 4 kb vector (Mlu I/Xho I).  The vector has been gel purified and dephosphorylated (shrimp alkaline phosphotase), the fragments ligated (NEB T4 ligase overnight at 16 C), and transformed into XL-1 supercompetent cells (Stratagene).   After plating/overnight incubation, very few colonies are present, and after screening for positives with Mlu I/Xho I, there is no insert.  Any suggestion/tips?


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