Hello everybody,
I have been trying to generate a KO using the recombineering technique designed by Dr Pentao Liu et al. I have already my BAC and I ensured that I had the correct one by PCR and sequencing. I have generated my first minitargeting vector with 2 homologous arms. However, I am stuck at the very first step of recombineering. I am using the SW105 strain to retrieve a 9KB genomic fragment from my BAC. I always seem to end up with my minitargeting vector each time. I have tried all the tests suggested by Dr Liu's protocols available online, and each time I seem to end up with my mintargeting vector. I am using homology arms of approx 250pb on each side should I use longer homology arms? Should I CIP my linearised minitargetting vector before performing the recombineering step?
