I am new to the CRISPR-Cas9 system and wonder if there is any good protocol for cloning gRNA into vectors. Given that they are quite small in size, is there anything that I need to be careful of when cloning them?
I decided to use three of the vectors from Zhang lab (https://www.addgene.org/crispr/zhang/) and I am going to use the protocol they suggested (https://www.addgene.org/static/cms/filer_public/04/43/04439a9b-fd21-419c-b872-426b7b0e122f/zhang-lab-general-cloning-protocol.docx).
I am going to start it next week and will let you know how that goes!!
Hi everybody! I have a question about Zhang lab protocol in which digestion/ligation reaction is made in the same tube. When BbsI cuts pDNA a fragment is released from the vector and then, gRNA are ligated. However, is possible that fragment released be ligated too?? Transformed bacteria in negative control (Without gRNA insert) could grow in LB plates?
Did you phosphorylate the annealed oligos? I am trying to clone a sgRNA into pX459 vector using Zhangs Labs Protocol, but at the end I do not have colonies.