Hello Mr. Burby,
sorry for the late response. I switched to pPB41 for Spacer cloning procedure. The first attempt to incorporate the Spacer seemed to be successful after gel electrophoresis, but the sequencing results said it´s not incorporated. Also the first Spacer and the second CRISPR Repeat disappeared in pPB41 sequence. I uploaded an picture of the pPB41 sequence before cloning, pPB41 with cloned Spacer made by SnapGene and the sequencing results. My professor told me, it could be possible that the regions got eliminated by homologous recombination due to the repeat homologous regions. Therefore i would like to ask whether you had the same problem and how much positive clones do you typical sequence after cloning.
Tobias Zumbusch 9/6/2018 5:12:50 AM Reply