How do I arm DNA


Cornel Mülhardt, author of the method knocker The Experimenter - Molecular Biology ”, philosophizes again about the everyday life of the researcher. Deep insights into becoming and passing away, the dialectic of haste and patience and the contradiction between the search for knowledge and the search for a partner are presented. If you have even deeper insights - be it those of dialectics or the agarose gel - write us a short comment. It will be printed in the next issue. Without annoyance from the reviewer! A unique opportunity to extend your list of publications.

As is well known, only calm is enough. Think. An example: It is Thursday, the end of work is approaching and you have an appointment to go to the cinema. The digestion of a few important clones that we have been working on for days now rest in the heating block. This under ever increasing pressure, because after all, the fate of the whole project depends on it. You could now look forward to the evening in a relaxed manner, mentally initiate the switch to wine, women and singing and sweep the workplace - if you work in the institute's own workshop. But if you are penetrated by the scientific spirit, you have to step up a gear at this point, because if you found the right clone, you could quickly set up a maxi culture and gain a whole day. So you drove an agarose gel in no time!


The question of the price, however, is: What is in a flash? How long does such an agarose gel take? Dissolving the agarose, pouring the gel, gel time, electrophoresis, staining and documentation can take two and a half hours in the longest version. Two and a half hour! Isn't that faster? It works.


Dissolve the agarose

To dissolve the agarose, you have to bring it to the boil. Is there anyone else who does this with a bunsen burner? Let it go. If microwave ovens have any reason to exist in this world, it is heating agarose. It's quick and clean. One minute of full power per 100 ml of liquid and then continue until the Chose just does NOT boil over. Yes, agarose boils over very easily and creates a mess.


Cool the agarose

If you have time, you can let nature run free. The hasty one will speed up the process. Putting the bottle under the open faucet is popular, but nonsensical, because the amount of water consumed is enormous and, contrary to popular opinion, drinking water flows out of the vast majority of laboratory taps. Better to put the bottle in a bucket or ice bucket. Speaking of ice, this is of course the fastest way to cool it, but ice cooling requires that the agarose is constantly mixed well, because agarose starts to gel at around 40 ° C on the bottle wall.

I was told that (some?) TA schools teach to acclimate the agarose in a 50 ° C water bath before pouring it. Dear TAs, refrain from doing this if you don't explicitly want to drive your boss crazy. Agarose can be poured at any temperature, starting at 99 ° C (which, however, the gel slides suffer enormously) down to the gelling point. Once you have poured the gel, you can accelerate the gelling process by placing it in the refrigerator - provided that the agarose has been cooled sufficiently beforehand, otherwise the refrigerator is transformed into a cloud chamber with an icy back wall. It's even faster in the freezer, but there are three problems:
a. there are hardly any flat surfaces in the freezer, which can be clearly seen in the gel;
b. you risk forgetting the gel and the agarose will freeze and become unusable; and
c. the icing problem is much bigger.

Anyone who is often faced with the problem of having to use another gel quickly should pour gels in reserve. Agarose gels will last forever in the refrigerator if they are kept moist. In individual cases it is sufficient to wrap the gel in cling film, otherwise you should get a plastic box that you fill with a little electrophoresis buffer (a level of 1 cm should be sufficient). If you can, you should keep the gels together with the gel slide, because the pockets tend to tear if handled carelessly; you usually only notice this when you load your DNA and the blue marker begins to spread under the gel. An alternative is the sheet cake principle: you pour a very large gel in the morning, from which you cut out the pieces you need during the day.


Electrophoresis

Applying the right tension to the gel is a feat. Because every gel apparatus is built differently, reference values ​​of 0.5-10 V / cm electrode spacing can be found in the specialist literature, depending on the electrophoresis buffer, fragment size and agarose concentration. In practice, these values ​​are of only moderate help. We used to have fun competitions in the laboratory to see who can run his gel at the highest voltage. A useful side effect was the knowledge that the gels start to melt at the top first, which gives you plenty of time to turn the tension back down. I can only recommend to every researcher who is pressed for time to playfully explore the limits of his equipment. This knowledge can be very useful in moments of extreme time constraint. One should know, however, that the resolution of large bands (from approx. 5 kb) suffers. If you want to separate large quantities of large fragments, you have to go the opposite way. It electrophoreses better at low voltage overnight (here, too, a few playful test runs are recommended before the emergency).




coloring

The classic method consists in staining the agarose gel after the run in electrophoresis buffer with 0.5 µg ethidium bromide (EtBr) / ml. This approach has its advantages, but it takes a lot of time (20-30 min for the staining and possibly another 20-30 min for decolorizing in water to improve the contrast) and experience has shown that it is a huge mess because the friendly colleagues with the ethidium bromide dripping gels make a pilgrimage across the laboratory. If you want to have fun, you should arm yourself with a UV lamp and pay a visit to such a laboratory in the evening, the reactions of colleagues who are more concerned about their health are very amusing.

It’s faster if you add the EtBr directly to the agarose when pouring the gel; Maniatis also recommends 0.5 µg / ml here, but a fifth of this amount does the same. Contrary to popular belief, EtBr is UV-sensitive, but it is definitely heat-stable, so you can easily prepare larger amounts of agarose and reheat the remains later without the EtBr suffering as a result. The advantage is that you can take out the gel at any time, look at it and let it continue running, the disadvantage that the ethidium runs in the opposite direction to the DNA. Somewhere in the middle of the gel, the EtBr front and the smallest DNA fragments cross each other, and from this moment the DNA becomes discolored again and fades. In many cases, those who need long running distances have to resort to the dye bath, or they also add EtBr to the electrophoresis buffer - another huge mess.




Summa

If you optimize all points, you can reduce the time for an agarose gel to up to 20 minutes in an emergency, short enough to start maxi culture and get to the cinema in time. Maybe it will work out with the neighbor. However, you should take a little more time for them.