it could be that the bacteria just overgrew (if you put them in the incubator in the early afternoon and took them out next day later in the morning). I would just pellet all bacteria and to mini prep from that.

Add a positive control to the growth experiment (e.g. a strain you know to grow in that media/antibiotic) and repeat it.

It is more likely to have a contamination in your materials (detergent on glassware, bad batch of bacteria...) than severe toxicity that is absent on a plate.

Also, there was no need to clone all fragments into TOPO and then each of them back into MigR1. If you have the full length MigR1 construct you can simply PCR out the bits you want to delete. That is done similar to mutagenesis PCR.

it could be that the bacteria just overgrew (if you put them in the incubator in the early afternoon and took them out next day later in the morning). I would just pellet all bacteria and to mini prep from that.

Try to shake with higher speed. Use sterile plastic tubes or flasks. Make sure the temp is 37 degree C. Good luck.

Does your vector have any viral recombination sites? If so, transformation your vector in another strain of E.coli other than TOP10

Hey Jim,

migR1 is listed as high copy number plasmid. i agree with above comment about over-growth. positive control of migR1 plasmid without any inserts can help deciding time-point for growth!

best,

Gurjot

Thanks for the input everyone. Overgrowth is not likely the problem based on previous experience with this same vector and positive control outcomes with MigR1. I believe the problem is likely with the Top10 cells (pointed out by Weitai), as MigR1 is a retrovirus that may not work with Top10 cells.  I will try the transformation into DH5alpha and keep you all updated.

I have no explanation but I have experienced similar problem for two genes I used for cloning. Other genes with similar cloning strategy, same cloning vector and competent cells did not have this problem, so in my opinion the problem is related to the specific gene(s). For one of the problematic genes I even had a different colony morphology on plate. Only way to 'solve' this was to grow the colonies in larger volume in erlenmeyer flask, repeatedly spin down in same eppie tube to collect enough biomass for the DNA isolation. In the next cloning step, this problem did not occur.

Hi Jim,

If you rule out fungal contamination of your cultures do they grow normally for a few hours and then just seem to crash? Strange growth patterns might mean 'phage-sometimes you see a crash and then a second growth phase as a few cells spontaneously mutate to combat the 'phage but they rarely still contain your plasmid or protein in any useful form. Closely monitor the growth curve then if it looks suspicious you should get hold of some 'phage resistant E.coli.

Colony morphology changes are also indicative of 'phage-do they look 'stellar' (bits missing from edges like a  chewed biscuit)  rather than nice and round?

The fact that it appears to be related to specific genes could be that the source of the  'phage was a plasmid template from an external source, so you problems appearing at the same that you began work on a particular gene may not be coincidence.

If it is 'phage you should also ask other users of your incubators if they have problems as it passes fairly easily from culture to culture and is really hard to remove from the lab environment, shakers etc.-the only option is to remove their 'host' by everyone in the lab switching to the 'phage resistant strains.

If you want to stick with your current strains you can probably make them 'phage resistant but they then require re-validating in your protocols so it may be easier to just buy some in.

Good Luck

Thanks everyone for the input.  It turns out that our new stock of amp had somehow lost some of its activity.  It was working on the plate to a very small degree (causing colonies to be smaller than usual), but then killed the bacteria after some growth in liquid.  With a newer stock of amp (and using DH5-alpha to avoid viral recombination and phage killing) the prep worked as usual. Although the issue wasn't one I expected it to be, after doing quite a bit of research into this I would recommend using DH5-alpha high efficiency or other competent cells as opposed to Top10 for cloning unless the plasmid has already been confirmed to work with Top10 (i.e. TOPO).

This is a couple of years late to the question but my best bet is that you are picking too much bacteria for initial growth. I've seen this many times when a new person enters the lab. They scoop up a good chunk of the bacterial pellet from the LB plate and swirl it around. This always results in stringy dead cells that foul of miniprep/maxiprep columns. Just touch your pipet tip to the bacteria lightly and touch the bacteria. Problem solved.

I was recently having the same problem. I solved it by changing the bacterial strain. TOP10 cells did not work, they'd grow in the agar, but would lyse in liquid culture. My plasmid was antiviral, and it's possible that there was recombination going on. I switched to DH5a, after also testing Stble3 and OminiMax. Now it's working.

Hope this helps.

Just to revisit this, I actually did figure out what had happened to these cultures. I wondered about cell specificity of this toxicity problem, and discovered that was not the problem in my case. We believe what happened was an issue with the antibiotic selection. Our antibiotics had partially degraded. For some reason, this partial degradation seemed to affect the selection on the plates more than in liquid culture, as the bacteria started to grow and then died, thus giving the lysed bacteria at the bottom of the culture tube. We bought new antibiotics, and the procedure was up and working again in no time!