Isotopic Labeling of Cyanobacteria and DNA Analysis
Prepare Pro99 , SN , SNAX or Amp1 medium according to directions , but use 15N ammonium chloride which will provide the heavy isotope .
15 NH4Cl is available from Cambridge Isotope Laboratories , Inc . ( # NLM - 467 - 1 ) .
Grow cyanobacteria in the medium with heavy nitrogen and transfer at least 3 times before use .
Harvest the bacteria grown in heavy nitrogen .
Extract bacterial DNA using standard methods .
Quantify the DNA using Quant - iT Pico Green .
Use at least 10 µg of DNA for density gradient centrifugation .
For density gradient centrifugation , a Beckman VTi 65 vertical rotor was used with 13x48 mm OptiSeal polyallomer tubes ( 4.9 ml capacity ) .
Mix the DNA with TE buffer ( 10mM Tris , 1mM EDTA , pH7.6 ) to a final volume of 0.9 ml .
Mix the DNA with 4ml of CsCl prepared in TE to a density of p1.8 ( measure the density of the final solutions , they should be p1.7 ) .
Dispense 4.9 ml of the DNA sample in CsCl into the OptiSeal tube and plug with the black caps .
Load the tubes into the rotor and put caps on all carriers .
Centrifuge at 44,000 rpm ( = 184,678.5 g ) in a Beckman L70 or L80 ultracentrifuge for 48 hr at 18°C .
Collect 0.2 - 0.25 ml fractions .
Calculate amount of DNA in each fraction using Quant - iT Pico Green ( perform in duplicate ) and measure the density of each fraction .
Determine the density of the fractions with DNA ( plot ng of DNA versus CsCl density ) .
