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Bellas ERL erl470160suppdata

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Bellas ERL erl470160suppdata
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  Bacterial carbon production methodology  Nine 1.5 ml sub-samples from each cryoconite hole were removed and transferred to sterile 1.7 ml microcentrifuge tubes. Incorporation was started by addition of 3 H-leucine (100 nM final conc.). Three of the tubes were incubated as killed controls by the immediate addition of 100 µl glutaraldehyde. The tubes (6 live, 3 killed) were incubated at in situ  temperature for 3 hours and incorporation was stopped by addition of 100 µl glutaraldehyde (2% final conc.). To precipitate the protein, trichloroacetic acid (TCA) was added to the tubes to a final concentration of 5%, (89 µL of 100% [w/v]) and left overnight at 4°C. The tubes were then centrifuged at 16,000 × g for 10 minutes and the supernatant aspirated. Samples were washed by addition of 1.5 ml 100% (w/v) TCA and vortexed briefly, before being centrifuged again for 10 minutes at 16,000 × g and the supernatant aspirated. A final wash was carried out with 80% ethanol as above. Ultima Gold™ (Perkin Elmer) liquid scintillation cocktail was added to the microcentrifuge tubes (0.5 ml) and the tubes were placed in scintillation vials before being radioassayed in a Tri Carb 2810 TR liquid scintillation analyzer (Perkin Elmer). To correct for incorporation in dead samples, disintegrations per minute (DPM) values from the controls were subtracted from the live samples to give disintegrations  per minute incorporated (DPM inc ). Bacterial protein production (BPP) was calculated from disintegrations per minute (DPM) using the equation:        (Simon and Azam 1989). Where mol leucine inc is calculated by:           Where SA is the specific activity of 3 H-leucine (17.3 Ci mmol -1 ); 100/7.3 = 100/mol% of leucine in  protein; 131.2 is the molecular weight of leucine; ID is the isotope dilution of 2 as used in Simon and Azam (1989). BPP was converted to bacterial carbon production (BCP) by multiplying by 0.86 (Simon and Azam 1989). BCP was converted to new bacterial cell production (NCP) by dividing BCP by a range of mean cellular carbon contents per bacterial cell (10.68  –   30.53 fg C cell -1 ) calculated from the biovolume.    RG2 a 051015202530    C  o  u  n   t  s   (   1   0    8    g   -   1    ) 0510152025 RG2 b 0510152025300510152025 RG2 c 051015202530    C  o  u  n   t  s   (   1   0    8    g   -   1    ) 0510152025 RG1 a Incubation time (hours) 051015202530010203040 RG1 b Incubation time (hours) 051015202530    C  o  u  n   t  s   (   1   0    8    g   -   1    ) 010203040   Supplementary Figure 1. Virus-like particle (VLP) and bacterial counts (BDC) in all virus production experiments. Three sub-replicates were incubated per independent cryoconite (Rep 1-3). Label denotes site, where there is more than one cryoconite per site a,b and c denote independent cryoconites. Note change of scale to accommodate higher counts from RG1a onwards. ML a 051015202530    C  o  u  n   t  s   (   1   0    8    g   -   1    ) 010203040Rep1 VLPRep1 BDCRep2 VLPRep2 BDCRep3 VLPRep3 BDC ML b Incubation time (hours) 051015202530010203040  AB Incubation time (hours) 051015202530    C  o  u  n   t  s   (   1   0    8    g   -   1    ) 010203040
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