Problems with overnights - (Jan/22/2013 )

Hello,

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?

Thanks!

You should worry that the antibiotics have degraded (they likely have). When it takes that long for your culture to grow, expression becomes very tricky. How will you grow your bacteria to a specific OD (unless you monitor the culture for 24 hours)? Anyways, try to see if you can retransform your plasmid into regular BL21 cells and repeat. If this fixes the problem, you just had bad rosetta cells. I have used both Rosetta pLysS cells and BL21 star DE3 cells for the same plasmid with nearly identical results for two proteins.

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?).

Ampicillin might be a problem, because it is not one of the most stable antibiotics. But chloramphenicol is (if you don't put it under UV light all the time). And I guys you grow them in an incubator. What you could also try is to lower the antibiotic concentrations and see if they grow better, you might have to optimise the growth with this particular plasmid.