We are studying the Crispr/Cas 9. We have clonned our gRna into pSpCas9(BB)-2A-Puro (PX459) V2.0. Now, we are planing to do cell line experiments. Our cell line is HEK293T. Our crispr vector is pSpCas9(BB)-2A-Puro (PX459) V2.0 . We have Puromycin Dihydrochloride GOLDBIO in the Lab. Let's start with my questions :) Firstly, for antibiotic selections, how much concentration and how puromycin we should apply ? Secondly, we are confused for clonal isolation part. We found different technique especially from the nature protocols, but we have not done any clonal isolation before in the lab, How can we achieve the clonal isolation easist and most efficiently ? Especially, the most confusing part for us the clonal isolation for now, can you explain the clonal isolation part in very detail :) ?If we can succeed these, than for functional testing , which method do you suggest ( Surveyor assay, t7e1 assay or any other than these ) Thanks for your help
Hey hey!
For your first question: we always use 3µg/ml puromycin to select for transfected clones (with HEK cells, the concentration differs with cell line). Just have an extra well with untransfected cells to see when all wild type cells (or nearly all) should be dead.
2. If you have access to a FACS-sorting device: use it! Most easy and efficient way of getting single cell clones. If not just use the dilution protocol explained in the nature paper. Therefore you plate your cells at a very low density. This means that you plate statistically 0.5 cells/well in a 96 well plate and hope for the best. Count your cells, add ~60 to 12 ml of medium and give 100 µl of this cell suspension to every well.
3. There are a lot of protocols out there for detecting editing events. I experienced that most of them are very costly for the small benefit they offer (namely surveyor). Try the protocol in this link if you like:https://www.ncbi.nlm.nih.gov/pubmed/25236476
You could as well just sequence your bands directly. Might be the easiest way.
Hey i have a problem with that plasmid, i can´t ligate the sgRNA. Can anyone tell me your proceedng to ligate the sgNRA in the plasmid digested??
Thanks
Use golden gate cloning is really easy to do it. Check the paper called sap-trap in c. Elegans there is an elegant and efficient method to do anything.
Hello,
I'am using this plasmid (PX459) with an induced pluripotent stem cell line (IPSc). I have a problem with my cell viability, a lot of cell death after transfection and even more after puromycine selection. Is it a problem of vector integration in my cells ?
Here's a general protocol for transfecting HEK 293T cells with the pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid:
Materials:
Protocol:
Remember to validate the efficiency of CRISPR/Cas9-mediated genome editing using appropriate assays such as PCR, sequencing, or functional assays depending on your experimental design. Additionally, optimize the transfection conditions and perform appropriate controls for your specific experimental setup.
l This protocol list might provide further insights to address this issue.