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There must be in this internet, some sites that actually match the molecular cloning theory with the practice and the tools, but I seem to fail to find them. So it is time to review what we had done in Bangalore, and put it together step by step.

In Part-1, what we did technically in making a glycerol stock of the arsenic GFP reporter bacteria is outlined. Part-2 will try to put this together in the context of the molecular cloning entire process.

From a stab culture to glycerol stock

We first revived an agarose stab culture that arrived by post. More on the bacteria is here.

This meant making the media (food for the bacteria) and pouring agarose plates (food with footing), and also learning what means to be bench sterile. Also, the fact that we use an antibiotic that selects for our bacteria (other bacteria from around should be killed off because they won’t have the resistance gene).

Taking a scraping of this stab culture, and expanding it by growing a small liquid culture overnight is well described in the Jugaad GFP site from previous years’ of (Art)ScienceBLR activities. The only difference with what we did, and what they did is that they started with a freeze dried (lyophilized) stock, and ours was a room temperature stock that didn’t need to be rehydrated.

 LB media

What we used was powder already mixed, but if making from scratch see the details in the openwetware protocol, In 1 liter of distilled water, mix the following, and autoclave (or pressure cook!) in a partially closed container.

LB agar plates – similar nutrients as above, use 1.5% agar. When cooled, add so that we have a final concentration of 50µg/ml kanamycin (freshly made).

Then, next morning, from the overnight small volume culture, we

(1) started a larger volume culture to get the growth phase bacteria for making glycerol stocks. Glycerol stock making is nicely described here. This is a way to keep a long-long term stock, at -80C.

(2) Plated dilutions of the overnight culture on LB agar plates with kanamycin 50µg/ml

But technique is only useful, when you know when how and why we do things this way. Knowing this is the beginning to hack / think outside the box.

Recommended reference beyond wikis (although they are great):

Addgene has a cloning guide with protocols.

Classic book on the techniques of molecular biology: Molecular Cloning, A Laboratory Manual by John J. Sambrook, David David William Russell.

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