The plasmid is quite big which might explain why it is difficult to transform bacteria (is it a lentiviral vector?).
If you could give additional details you might have a chance to have improved answers to your question.
Hello, I suggest you follow this protocol:
At step 7 instead of this "Add 250-1,000 μl LB or SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min. "
Do this " Add 250 μl LB or SOC media (without antibiotic) to the bacteria and grow in 30°C shaking incubator for 2 hours. Do not shake too hard, gentle motion might be fine (500 rpm).
At step 9 instead of this "Incubate plates at 37°C overnight."
Try to incubate at 30°C. I would even suggest to try to grow them at lower (25°C, or 16°C) temperature if you can prepare a second plate.
It will take much longer than overnight to grow the cultures, probably more 48 hours (might be 72 hours at 16°C).
Hope that helps good luck!