Problems with overnightsOvernights
Posted 22 January 2013 - 06:22 AM
I currently cloned in a 5kb gene into the PGEX vector (5kb), so now my current plasmid is 10kb. After cloning, I transformed the plasmid into stellar competent cells to create a DNA library. I then did a miniprep (and also confirmed by restriction digest that my insert was still there), then I transformed the plasmid into Rosetta Competent Cells (which are BL21 derivatives) and here is where my problems are starting.
So I've come to discover that it takes about 48 hours for my overnights to grow. This also happens when I finally do get a 5mL overnight to grow, then transfer that into a 50mL overnight. I'm worried that the bacteria that start growing around 48hours are growing because the antibiotics have degraded by this point (Ampicillin and Chloramphenical).
Does anyone have any experience with this? Is 48 hours to grow an overnight sound about right? Also why can an overnight of my plasmid in Stellar Competent cells grow "normally" in an overnight, but the plasmid in Rosetta Competent Cells takes forever to grow?
Posted 22 January 2013 - 07:42 AM
In short, something is not right since the stellar cells grow fine. I doubt the gene is toxic since your normal competent cells grow fine. Just make sure your transformation into the Rosetta cells worked (are you 100% sure your plasmid is in there?).
Posted 20 February 2013 - 04:41 AM