STABLE ISOTOPE PROBING (SIP)

Stable isotope probing (SIP) is based on the observation that DNA molecules of different densities can be separated by ultra-centrifugation in a concentrated solution of cesium chloride (CsCl). CsCl density gradient centrifugation has a long history and was first used to show the semi-conservative nature of DNA replication in one of the most elegant experiments in biology (Meselson and Stahl 1958). In Messelson and Stahl’s experiment, E. coli was grown in medium in which all available forms of N contained the heavy, stable isotope 15N. 15N is chemically identical to the more common stable isotope of N, 14N. However, fully labeled with 15N, DNA has an average density of 1.722 g cm-3, whereas 14N-containing DNA has an average density of 1.700 g cm-3 (Buckley et al. 2007a; Buckley et al. 2007b). Similarly, 12C-containing DNA has an average density of 1.700 g cm-3, whereas its fully labeled 13C counterpart has a density of ca. 1.730 g cm-3. These small, yet significant, differences in density can be exploited during centrifugation under extremely high speeds (70-90,000 g) in a 5.6 M CsCl solution, which has a density of 1.700 g cm-3. The high g-forces result in a density gradient as Cs+ ions migrate towards the bottom of the centrifugation tubes. Ethidium bromide can be used to visualize the heavy and light DNA, which form distinct bands visible under UV light and influence the buoyant density of DNA. An example of this is shown in Figure 2B. CsCl density gradient centrifugation was more recently applied to study microbial communities (Radajewski et al. 2000). The goal of this groundbreaking work was to link specific processes in a microbial community to the identities of the bacteria performing them. The authors show that 13C-labeled DNA is generated by a population of bacteria growing on a 13C-enriched substrate and that this 13C-labled DNA can be separated from 12C-DNA, contained in bacteria that do not grow on the labeled substrate. The isolated DNA was then taxonomically characterized using routine molecular ecology methods. The combination of these techniques (incubation with a labeled substrate, CsCl density gradient separation, and phylogenetic analysis) is now referred to as ‘stable isotope probing’ (SIP). Radajewski et al. (2000) demonstrated that SIP is a powerful technique that allows for the taxonomic identification of microbes performing specific metabolic processes under in situ conditions (Radajewski et al. 2000).

Generate CsCl stock solution by adding the following to a sterile 50cc tube:

CsCl Stock

• 12.95 g CsCl
• 10 ml Buffer A (see below)
• 1.2 ul Ethdium Bromide stock (10mg/ml)

Don't add too much ethidium bromide, since background fluorescence can easily overwhelm the fluor of the DNA you are trying to see, especially if you don't load tons of it. Check the density of the solution with a refractometer. It should be around 1.403.

For a 13C gradient add the following to a polyallomer tube:

• 4.45 ml CsCl stock
• 0.30 ml DNA solution in Buffer B

Cap tubes and invert 20-30 times to mix. Then balance your tubes extremely well (delta<0.001) by removing liquid from the heavier tube ! (remember that the density if about 1.4, so each ul is about 1.4mg) .

When you are done balancing, weigh your tubes again, then weigh them again to make sure your tubes are balanced.

I then use a VTI65 rotor to spin my tubes at 40,000k (ca. 180,000 g) for 48 hours. Visualize on Transilluminator. I find that this separates 13C labeled DNA nicely from unlabeled DNA.

• 15 mM Tris
• 15 mM KCl
• 15 mM EDTA pH 8.0

• 50 mM Tris
• 15 mM EDTA pH 8.0