pCAMBIA Construct

The main construct in the 2015 iGEM project is the Cas9 - pCAMBIA plasmid. We had many difficulties when trying to ligate the pcoCas9 and/or our Cas9 gene modified with restriction enzymes into a stock sample of pCAMBIA 654/766; pcoCas9 needs to be isolated, PCR amplified, and ligated into a digested pCAMBIA vector. Our PCRs were smeared when we verified on gels, possibly from shearing before running on the machine. We tried to PCR with less DNA and found this was more effective. Upon ligation we found that transformation was unsuccessful both using heat shock and electroporation methods. New primers were used to attempt to better isolate and amplify PcoCas9, and a diagnostic PCR was implemented in which various buffers and temperatures were tested in order to optimize amplification. New primers and a new plan with buffers and temperatures allowed for easier extraction of Cas9RE.

PCRs of Cas9 genes for Gibson assembly resulted peculiarly with a band that was just off in size, being 4kb instead of 5kb. We found some information on AddGene that insinuated that 5kb is possibly a valid size for our PCR product - however this product is not viable to be cloned into pCAMBIA. The isolation of Cas9 genes on PCR would still be successful and allow us to begin Gibson assembly work and begin attaching parts together for the sgRNA swap.

Attempts to acquire the pCAMBIA background appeared to be more successful when cutting with BamHI. This success was not repeated.

After working from various angles, PcoCas9 and pCAMBIA were digested using EcoR1 and Xba for a ligation.