I left the field of protein biochemistry to pursue a project in synthetic chemistry some years ago, and now this project is veering into protein expression and purification. We ordered two plasmids from Genscript, and they arrived in the form of what appear to be stab cultures. Therefore, we need to move forward with deliberate speed. I have a basic understanding of sterile technique, but my skills are rusty. What are reliable sources of information regarding handling of E. coli?

Now, we need to store our strains, which arrived as stab cultures. In the future, we may need to work out details of the purification of our enzyme, because it is coming from different organisms versus previously reported one. I have a good knowledge of classical protein chromatography but not medium-pressure chromatography. How should I get up to speed in this area? I realize that my questions are broad.

Edited May 20May 20 by BabcockHall

Regarding sterile techniques: It is probably not going to help you, but I always found in-person training way more reliable than books. The most important elements are proper setup in the lab (i.e., having lab space with no traffic or air flow from ducts etc.), deciding on basic technique options (e.g., working on flame vs biosafety cabinet) and then developing workflows that you are able to do comfortably that minimize contamination risks (e.g., develop a routine how to lay out your tools, samples, plates etc.).

There a lot of manuals and also free books that illustrate basic techniques, but again, I find that while they can be decent intros, almost all of them have gaps, which is understandable as the implementation is very location, workflow and skill-dependent. Each lab usually develops their own SOPs and lab culture based on their specifics (and sometimes we have weird quirks that people do but their meaning is lost in time. Fascinating, really).

Regarding storage, stab cultures can be stored at 4C for a week or two, but you should make multiple freeze cultures as soon as possible. I think you can find some basic protocols on ATCC, but what we routinely do is starting from dilution streaking, then pick a single colony and cultivate it in appropriate medium (e.g. LB+antibiotic for a given plasmid) overnight. Culture are then mixed with glycerol (25%-50% (v/v)) and frozen. For longer storage, DMSO methods are preferred.

For chromatography, the methodology is similar, and there are a host of other methods which might or might not be applicable to your needs. If you have a choice of methods I would just grab either a basic bioanalytics book and skim it, to just get an idea what is out there and what might work for you and then talk to multiple manufacturers and applications specialists to discuss what might work on your budget.

Alternatively you could approach from it a protein purification side. Importantly, I suggest you find folks to ask questions. This is one area I regret a bit as I learned being a bit too be self-reliant as grad student, which helped me a lot way later in my career. But if I had done the opposite, my projects would have proceeded much faster.

CharonY,

Thank you so much for a thoughtful reply. For better or worse, I have to be the teacher in this project. We poured LB-kanamycin plates today, and it seemed to go reasonably well. We will try streaking plates tomorrow.

BTW I found this video on pouring plates to be a helpful reminder to me, and I plan to look for more such videos.

Good luck! One thing I cannot stress enough is to clean up the bench and only have stuff you need at the moment- removing clutter just reduces so many points of failures. For plates, I usually do a full stack in one go, as if you keep the bottle tilted (rather than putting it down to grab a new stack) you tend to introduce less bubbles. But YMMV. Also, especially at the beginning be slightly paranoid about contamination and make extra dilution streaks to at least visually confirm purity (or use a microscope if you access to and experience with it).

When you use a metal loop, do you put a curve or bend into it. I found myself digging a little bit into the agar as I was streaking.

Does anyone sell electrocompetent cells for transformation by electroporation? Or is it just better to make one's own?