Hi all,
So I am continuously experiencing a very weird phenomenon (see images attached). I am trying to get E.coli DB3.1 cells competent together with SURE and XL-Blue cells. I always streak out the glycerol stocks of each on LB plates with relevant antibiotics, pick a colony for an overnight 5ml culture at 37C with the relevant antibiotics (tetracycline 15 ug/ml for XL-blue and SURE because I want to maintain the F-plasmid), and then inoculate overnight 1/100 in 120ml LB (10g peptone, 10g salt, 5 g yeast extract per 1 L media) in 200ml Erlenmeyer flasks. The cells grow fine to an OD of 0.3-0.35 in about 3 to 3.5 hours whereafter I put the flasks on ice for about 30 min to stop growth. When I spin them down (10 min at 3000 rpm), the pellet looks see-through/lysed and flaky and does not stay attached at all. The cells don't look like they are alive. Has anyone else experienced this? How could the cells be dead if the OD600 is increasing? I have been making competent cells for about 6 years and this has never happened to me. What am I doing wrong?
This is the initial spin-down from the liquid culture? If the cells were growing then they were alive. Have you tried streaking out a few of the pelleted cells? That would be a quick viability check. Does the broth look gooey or cloudy? If not, then you probably just have a funny pellet but the cells would be ok.
Anything new in the lab? New source of 50 ml tubes, different centrifuge? New type of cells?
I agree that the pellet looks odd, is this a fixed angle rotor? I'm using to seeing one at the base (we have a swinging bucket rotor).
In the end, if the cells are competent then that's what matters. Hopefully yours is a quick protocol. If not, here is one to try
Hi all just to clarify, so I have literally checked what is growing under the microscope and in comparison to the overnight there is absolutely nothing. When I streak out the pellet on LB agar plates nothing grows either. My issue happens when transferring the O/N to the flask to grow to the required OD, no treatment with CaCl2 yet. I am running a few checks today, and whilst the cells are growing to OD, I am spinning some of the cells down and this is happening again, although the OD is still increasing hourly as one would expect. The cells in the flask will not stay pelleted at any centrifugal speed, I have tried that. I have been using a fixed angle rotor, but have tried a bucket rotor with the same results. When I try to pipette the cells out the pellet lifts and I also can't decant without dislodging the pellet as well. I have no idea what is going on. The O/N is grown in exactly the same media that I am using in the flasks and it is looks like media that is inoculated when growing i.ei, cloudy in comparison to the uninoculated control that I am spinning in the incubator as well to rule out contamination. I have been using different glycerol stocks of bacterial cells and they all do this. I am at my wits end.
What kind of tubes are you using for the initial centrifugation step? Sounds like your cultures are growing well, and that different sources of cells are all having this pellet not sticking/forming issue.
Maybe try a few different sources of tubes? Like 50 ml conicals vs round bottom? Or maybe it's a centrifuge issue?
Even putting 1 ml in a 1.5 ml snap tube and spinning for 1 minute in a benchtop centrifuge should give a nice formed pellet.
What happens if you streak out cells from your culture flasks? Those sound like they are growing well but it would be another way to check.
Random though - any chance the tubes are coated inside?
Sounds horribly frustrating!
- Badges
- Science topic