So, previously, I pointed out some of the difficulties involved in getting reagents and other scientific things to a place like Nigeria. Anyway, I thought a post like this might therefore be useful for the odd reader out there who also does this sort of thing.
Because the norm is traveling by bringing stuff along as "carry on," here's a run down of what I know. Please add more in the comments section if you have a trick or two yourself.
1. DNA constructs: Basically, the easiest thing to transport. Should easily survive any trip you plan to make with it. Of course, spiking with EDTA will help, or just using the standard Tris/EDTA (TE) buffers.
2. E.coli clones: We're talking innocuous, non hazardous varieties here. This too is also pretty easy. Conventional ways of doing this are to spot a piece of filter paper with your culture and seal in a bit of plastic wrap. Should be able to survive the trip in this regard. If you are "freezing" the bug stocks, then having the culture in a 50% glycerol stock will be required.
3. Proteins: This will vary greatly, since proteins themselves vary so much. But this is where, the info is particularly handy (I think).
Antibodies: This will depend on the antibody - shouldn't be a problem though.
Restriction Enzymes: Here's a surprise - a lot of the common ones are hardier than you think. There's even a paper on this aspect, which you can check out here. Basically, it suggests that:
The stability of restriction enzymes as supplied by manufacturers without any modification has been examined. No reduction in activity was observed for three enzymes (HindIII, EcoRI and Tsp509I) held at ambient temperature or 4 degrees C for the period of study (12 months), while activity was observed for up to 12 weeks after storage at 37 degrees C, which was considerably better than following desiccation with trehalose, a recognized preservation technique. A larger trial of 23 different restriction enzymes held at room temperature for one week showed that all enzymes retained significant activity. As a practical demonstration of the usefulness of this finding, enzymes were posted to Africa by conventional mail (cost $1 US) and shown to retain activity upon arrival after three weeks in transit (compared to a cost of $1000 US by cold-chain transportation). Supplying enzymes to third-world markets should now be possible by removing the necessity for cold-chain transport. After arrival, enzymes can simply be stored in a standard domestic refrigerator.
How cool is that?!?
Heat Stable Polymerase: (speaking of "cool") In principle, the polymerase with the higher half life should be the most stable, but there isn't any specific data out there to test this (i.e. put your preferences towards a polymerase sold for it's ability to go for higher number of cycles than usual). As well, there appears to be a special Taq available that is specifically marketed as one capable of ambient temperature transport (Thermoprime Plus DNA Polymerase). I'll be taking this one this time around, so will let you know how it faired.
One other thing (with respect to the polymerase), is to take advantage of the lack of patent protection for the unaltered Taq Polymerase (all the ones that are commercially available are essentially modified forms of the enzyme). Specfically, if you can get hold of an e. coli bug containing the pTaq plasmid. This is basically a protein expression construct, so that the e.coli can churn out copious amounts of the enzyme. Because, e. coli bugs are much easier to transport, one option is to take the bug (which happens to make the enzyme). AND the best part of all this, is that purification of this Taq polymerase is the easiest thing ever - in essense, you boil, you spin, you keep the supernatent. Enzyme is in the supernatent, and the only thing that is conveniently functional because of the boiling step. How clever is that?
T4 ligase: This is a tough one, and still unproven. Apparently, a number of studies have shown that you can significantly extend its shelf life by spiking the reaction with 0.05mg/ml (basically no loss of activity over a 20 hour period at room temperature). Presumably, the same stability attribute can be conferred with the stock itself. Perhaps the enzyme stock is already at a high enough concentration to avoid these chaotropic effects, but I guess we'll see...
Reverse Transcriptase: There doesn't seem to be any data out there on stability after prolonged exposure to ambient temperatures. But there is data out there on heat stable DNA polymerases, who happen to also have inherent RT activity. For example, this patent (filed in 1994) has some useful information:
All reactions were mixed on ice and then incubated at 65C. for Taq. After 20 minutes the reactions were chilled on ice (0C.) and EDTA was added to each reaction to a 10 mM final concentration. A sample was removed from each reaction to measure alphaP32 dCTP incorporation.
RNA template + 0 mM MnCl2 (9CPM)
RNA template + .5 mM MnCl2 (256CPM)
RNA template + .7 mM MnCl2 (3088CPM)
RNA template + 1 mM MnCl2 (3977CPM) (this doesn't look too bad)
RNA template + 2 mM MnCl2 (2696CPM)
RNA template + 6 mM MgCl2 (737CPM)
minus template + .7 mM MnCl2 (5CPM)
minus template + 2 mM MgCl2 (3CPM)
DNA template + .7 mM MnCl2 (194,199CPM)
DNA template + 2 mM MgCl2 (209,559CPM)
Which means in principle, I could do a reverse transcriptase reaction with the Taq polymerase I'm bringing along.
Anyway, there you have it. Any other tricks would be much appreciated.
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I've been trying to come up with some simple ways to extract and preserve DNA myself, in the context of getting fresh, stable samples of bacterial DNA in the field with a minimum of steps. The simplest reliable (...as far as I know...) method for preserving DNA itself seems to be plain old dessication. Precipitate your DNA out with pure 2-propanol or ethanol, spin it down, then dump the alcohol and dry the tube. Alternatively, you can spot the DNA directly on to bits of paper (if you get an easily-dissolved variety of paper, you can apparently directly stick the strip of paper into, say, a PCR reaction without any further purification and have it work fine.) The blog post I linked to above mentions a couple of references to this technique.
I still wonder if it would be feasible to switch to a nice spore-forming organism (e.g. Bacillus subtilis) for transformation with plasmids. I still think E.coli is sort of the "Microsoft" of microbiology (this is not intended to be read as a good thing...).
In any case, I just think some nice sturdy dried B.subtilis spores containing the plasmids desired would store and travel a lot better than E.coli does. One of these days I'll have time to do proper experiments and see if I'm right about that. (If anyone has a surplus copy of the ~$400 apparently-out-of-print book on Bacillus protocols that they want to get rid of, I'd happily take it off their hands).
Good to know about the Taq polymerase production though - I've always wondered how much purification was required to get a useful product. Sounds like it doesn't take much at all. One less barrier to building my own operational "Hillbilly Biotech" lab one of these days...
Thanks for the info. This is useful!
For proteins, freeze-drying is generally the best method of long-term storage and should usually be the best form to transport them in.
The stability of restriction enzymes has interesting evolutionary causes. These enzymes may have evolved as toxic parts of so-called "addiction modules" (Googling this will find a review). These modules consist of two genes usually carried by genetic parasites such as plasmids. One gene specifies a toxic protein or RNA and the other an antidote to the toxin. For restriction-modification systems, the toxin is the restriction enzyme, which cuts host DNA into pieces unless the DNA has been protected by the methylase protein.
Natural selection acting on such plasmids has favoured addiction modules whose toxins are much more stable than their antidotes, so any cells that manage to get rid of their plasmid will run out of antidote and be killed by the toxin. When the addiction module is a restriction-modification system, cells that keep their plasmid are protected by the methylase, but cells that lose it die because the methylase is much less stable than the restriction enzyme.
ANnd that explains why methylases are so unstable (and expensive) but restriction enzymes are so stable (and cheap). (This explanation for the evolution of restriction-modification systems was put forward by Ichizo Kobayashi. I think it's brilliant)
Do you really need 50% glycerol to freeze E. Coli back? Or are you talking about freezing back broths? It has been my experience that enterohemorrhagic E. Coli doesn't even need any glycerol at all, just scrape back a lawn off of a plate into 250-300uL LB broth and it's good again later. To the best of my knowledge, microaerophilic bacteria need glycerol much more, and they're often still viable after freezing with only 20% glycerol in BB broth.
Is it possible to transport pre-made reagents/solutions like Skirrow's Supplement or Sorensen's Buffer? Or do they skew under transport conditions and thus necessitate on-site manufacture?