I have been working with CRIPSR-Cas system and using nucleofection method for plasmid delivery. I am co-transfecting with10 kb size CRISPR-Cas and 3kb size gRNA using Lonza amaxa 4D-X system. The cells are suspension B cells. Buffers for transfection are company SF buffer and also homemade buffers of which recipes obtained from the literature ( https://doi.org/10.3389/fbioe.2016.00099 ).
The problem is nucleofection with 3 kb size using lonza programs and tested buffers are OK but when I try them with large size plasmid I get max up to 10% ( very bad in my opinion). I need your suggestions, experiences! How do you successfully deliver your plasmids to cells apart from using lentivirus vector. I think this step is the most challenging part. Are there any tips and tricks? Does any of you experience with Lonza transfection or any other nucleofection method? What is your the most optimal transfection efficiency percentage to reach for a good editing efficiency? Any input is welcomed and appreciated!