I have been preparing competent dh5alpha cells in the lab with good competency not excellent. however, have not been able to transform my CRISPR plasmid yet. I am following all the desired steps still unable to attain the correct colonies. plz, throw some light where I can be making mistakes. Plasmid is from addgene (pSpCas9(BB)-2A-Puro (PX459) V2.0)
Hi Nupur, could you share some more information such as: Are you getting no colonies or are there colonies but they're not producing your plasmid? Which selection agent are you using? Have you taken any steps to validate the integrity of your plasmid? Are you using heat shock or electroporation?
I've never noticed any differences between the PX459 vector and any other plasmid when I've transformed it. I usually used the NEB5 alpha cells purchased already competent from NEB.
If your CRISPR vector is lentiviral based, I think it is better to use other strains of component cells instead of DH5a. It is easier to acquire undesired plasmids in this scenario.