Methodological details for conducting both DNA-SIP and RNA-SIP are given below. In general, the steps for performing SIP are independent of the isotope used, so the protocols below can be used for processing samples from any DNA- or RNA-SIP experiment. For DNA-SIP, a protocol including a secondary centrifugation step in the presence of bis-benzimide is also detailed as an optional deviation from the standard DNA-SIP protocol. Although this method is considered outdated by now, it is provided here for completeness. Since DNA- and RNA-SIP protocols share many similarities with each other, much of the protocol is given for both methods together, and deviations for each specific method are highlighted. All protocols assume that an environmental sample has been incubated in the presence of a 15N-labelled substrate and that total DNA or RNA have been extracted from the sample following incubation. Methods for extracting DNA or RNA from environmental samples are well established and go beyond the scope of this chapter. Many commercial kits exist for this purpose, depending on the type of sample, and also general-purpose lab protocols (e.g., \citealt{Angel_2012}).

Materials

Gradient preparation

  1. An ultracentrifuge, capable of achieving 177,000 \(\times\) g and equipped with a vertical or a fixed-angle rotor for tube volumes of 2--8 ml (typically 5--6 ml; e.g., VTi 90 from Beckman Coulter)
  2. Suitable polyallomer ultracentrifugation tubes and caps (one for each sample, e.g., Optiseal 4.9 ml)
  3. Refractometer (typically, Reichert's AR200 digital refractometer) 
  4. DNA sample in TE or water (0.5--5 µg; for DNA SIP)
  5. CsCl solution (prepare a 7.163M CsCl solution by dissolving 603 g CsCl in 500 ml of filter-sterilised molecular-grade water; confirm that the density is ca\(.~\)1.89 g ml-1; store at RT; for DNA SIP)
  6. RNA sample in TE or water (500 ng--5 µg; for DNA SIP)
  7. CsTFA solution (ca\(.~\)2 g ml-1; store at 4 °C; for RNA-SIP)
  8. Hi-Di™ Formamide (Thermo), or any other deionised formamide (for RNA-SIP)
  9. Gradient Buffer [GB; prepare a 0.1 M Tris-HCl (pH 8.0), 0.1 M KCl and 1 mM EDTA in RNase free water, filter-sterilise (0.1 µm) into clean glassware and autoclave]
  10. One 50-ml tube per gradient
  11. RNase free water (for calibrating the refractometer)
  12. Bis-benzimide (Hoechst 33258, 10 mg ml-1 solution; for DNA-SIP using bis-benzimide)

Gradient fractionation

  1. Refractometer
  2. 1.5 ml non-stick tubes
  3. Test tube utility clamp mounted on a stand
  4. 20 ml syringe
  5. A flexible tube (approx. 30 cm; for instance an elastic HPLC tube) attached to the syringe with a Luer-Lok connection fitting, and with an additional Luer-Lok connection fitting for a disposable needle
  6. RNase free water for displacing the gradient solution (enough to displace the entire volume of an ultracentrifugation tube times the number of gradients)
  7. Variable-speed, automatic syringe pump 
  8. Disposable needles: 23G and 26G

DNA-SIP fraction precipitation

  1. GlycoBlue™ Coprecipitant (15 mg ml-1) or molecular-grade glycogen.
  2. PEG 6000 solution (prepare a 30% PEG and 1.6 M NaCl solution by dissolving 150 g of polyethylene glycol 6000 and 46.8 g of NaCl in molecular-grade water to a final volume of 500 ml. Filter-sterilise and autoclave. Final solution is 30% PEG 6000 and 1.6M NaCl)
  3. EtOH (70%; molecular grade in molecular-grade water)

RNA-SIP fraction precipitation

  1. GlycoBlue™ Coprecipitant (15 mg ml-1) or RNA-grade glycogen.
  2. EtOH (100%; molecular grade)
  3. Na-Acetate solution (3 M; pH 5.2, RNase free)
  4. EtOH (70%; molecular grade in RNase-free water)
  5. Optional: RNA Storage Solution (Ambion)

Methods

Gradient preparation

  1. Prepare all solutions in advance.
  2. Equilibrate the CsCl (for DNA-SIP) or CsTFA (for RNA-SIP) solution to room temperature for about 60 min (if stored at 4 °C).
  3. Calibrate the refractometer using pure water.
  4. Prepare the gradient mixture depending on the type of SIP (see below):
For DNA-SIP:
  1. For each gradient, mix GB, DNA sample (0.5--5 µg) and CsCl solution to reach the desired density (typically 1.725 g ml-1) in a separate 50 ml tube. The volume of CsCl solution needed to achieve a specific density is given according to equation \ref{eqn:mix1}.