Cloning: Where to Hit The Pause Button

Here are a few hints on where you can pause in your cloning experiments while working on other projects:

Restriction digests can be left at room temperature over night over even over the weekend. Prolonged digestion occasionally results in star activity, so be aware of this possibility if you encounter subsequent problems with the DNA fragment.
Gel extraction of DNA from an agarose gel can be put off indefinitely. Try storing the gel slice in the fridge overnight, or even melting the slice in buffer and freezing it at -20°C or -80°C. Degradation of the DNA will occur at the same rate and under the same conditions as soluble DNA, so use a colder temperature for longer storage.
Ligations can be done at room temperature or cooler (think 12-16°C) overnight or even for a few days, if you’re really busy. You can also store a ligation in the fridge and take it out later to continue ligating at room temperature for as long as necessary.
Transforming E. coli is usually an overnight procedure. If you incubate the plates at room temperature, however, colonies will appear three days after plating, instead of the usual one day at 37°C -perfect for sneaking in one last experiment on a Friday, without having to come in over the weekend.
We sent a sneak peek of this article to our newsletter subscribers and invited them to add their own tips. Here are a few great ones from John Mackay:

You can keep DNA extraction samples in the fridge once you have added 1) the extraction buffer (e.g. GITC or CTAB) or 2) chloroform (prior to spining) or 3) ethanol for precipitation.

For cloning, PCR reactions are stable in machine over weekend… without a 4degC hold – that’s a good way to bust your machine. I have left reactions on bench for several days before purification etc, although I wouldn’t suggest it for T/A cloning – fresh product is best.

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