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Background: I have previously worked with RNA, and then we use laminar flow hoods for any work where we do plating or inoculation of cultures while applying sterile technique. I am now working in a biochemistry lab. I'm told that I can work on a lab bench, while doing basic cloning for production of plasmids in E.coli, and don't need to use a Bunsen burner as long as I apply sterile technique (and in most cases I don't see people sterilizing the bench with enthrall prior to work). However, if I do any work which relates to expression of proteins - then I should use the Bunsen-burner while applying sterile technique.

Question: Is it really so that you don't even have to use a Bunsen-burner while plating (normal E.coli: XL1-blue cells)? in this case, why? Are there not always something in the lab which can grow on kanamycin plates? We don't use white/blue screening, we simply spread XL1-blue cells onto LB plates with kanamycin - then pick a averaged sized colony to make a LB-culture. It seems to me that we are just lucky that we usually pick the right colony (which we do confirm by sequencing after mini-prep).

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    $\begingroup$ As answered you can do without the flame just fine for stuff that you do not want to store for an extended period of time. In my experience if you open a kanamycin plate for a couple of minutes, only 1 in 10 plates will have some kind of contamination, almost always some sort of fungus. Yes, there's stuff that will contaminate your plate, but there's just not that much. $\endgroup$
    – VonBeche
    Jan 13, 2017 at 16:50

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Short answer is it never hurts to practise thorough aseptic technique.

My general rule of thumb is, if I'm opening a culture of bacteria (plates, eppendorfs, tubes etc.) I do it under a bunsen - the exception being if I plan to lyse the cells anyway.

For protein purification, sterile technique is important if you plan to store the sample for a while. Proteases in the environment and on your sample/self can make short work of destroying all your hard work.

Never trust your antibiotics fully. For one thing, they often won't stop fungi, which will ruin your samples even in the presence of some pretty nasty chemicals. If you're using some of the crappier antibiotics (Ampicillin is a prime example), the antibiotic degrades over time. B-Lactams in particular are an issue, as the B-lactamase is secreted in to the media. By day 2 of a culture, there will basically be no ampicillin left!

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    $\begingroup$ The ampicillin is a very important point. I once had to train some people who always had their maxiprep fail. After going through every step many times, I saw that they had made many frozen solutions of ampicillin and kept them at -20 for 6 months or more. I never use frozen ampicillin older than 1 month, keep it as dry powder until you need it. $\endgroup$
    – user137
    Jan 13, 2017 at 16:57
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For most plasmid preparation, strict sterile technique is not necessary. The antibiotics in the media will select for bacteria which carry a resistant plasmid, so you don't usually have to worry about other organisms getting in.

However, when you're using several types of plasmid, you do need to be careful to avoid cross-contamination, because each plasmid is resistant, so bacteria transformed with plasmid A will live just fine on plasmid B's plate. When plating your cells, be sure to pass your cell spreader through a flame. It doesn't have to be an official Bunsen burner, I used an alcohol burner just fine.

If you make a big bottle of media and use small aliquots at a time, then the bottle should only be opened inside the hood.

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  • $\begingroup$ that sounds rational, however, how about protein expression? because you also use antibiotics there, but I have heard that e.g. bacillus is a very common lab bacteria that a gets everywhere and grows quite fast even with antibiotics? I mean, there is a good reason why media is autoclaved before use even though you use antibiotics. Why should I not take the same consideration with protein expression using e.g. rosetta as with basic cloning using XL1-blue ? $\endgroup$
    – CuriousTree
    Jan 13, 2017 at 15:29
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    $\begingroup$ @CuriousTree I've never done much protein expression, but I think part of why we can get away with less than ideal aseptic conditions with plasmid prep is the time. We never let the bacteria grow longer than 24 hours, then we lyse and purify the DNA. Longer than that and the antibiotics aren't reliable. How long are your protein cultures grown for? Do you keep the cells frozen? Do you plan on thawing them out and starting new cultures with them? $\endgroup$
    – user137
    Jan 13, 2017 at 17:02
  • $\begingroup$ degradation og antibiotics is a good point, we normally grow the cell from the morning then express our protein overnight before harvesting, and don't re-culture them. All in all they grow about 24 hours, in presence of kanamycin and chloramphenicol. $\endgroup$
    – CuriousTree
    Jan 13, 2017 at 19:59
  • $\begingroup$ That's only true if Plasmid A and B are different but contain the same basis of resistance. A kanamycin plasmid won't survive on chloramphenicol and vice versa. $\endgroup$
    – Joe Healey
    Jan 13, 2017 at 20:21
  • $\begingroup$ @JoeHealey There aren't that many antibiotic resistance genes, so it's common to have two different plasmids with the same gene. The labs I have been in also tend to use the same vector for all our genes, so they all have the same resistance. $\endgroup$
    – user137
    Jan 14, 2017 at 14:55

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