Hi, I recently encountered some problem with my subcloning - I PCR the CDS of mCherry and Venus and inserted them into a 7kb vector to replace the EGFP. The insertion is 5' to a loxP-Cre-loxP casette, and the molar ratio of insert:vector=3:1. The PCR products were first TOPO cloned then released by RE digest (PacI-NotI) and then gel purified with the Qiagen kit. I then did the ligation 10 min @ RT using the NEB T4 ligase....and transformed into Invitrogen's OneShot TOP10. I got several clones for mCherry without any problem, but for the Venus subcloning all the clones I picked were showing extensive level of rearrangement/recombination.......does anyone know what might be going wrong in my hands?
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