Monica Stegman
June 14, 2011
MO Sampling
All direct counts:
For all direct count samples, fix 100 mL (90 mL water, 10 mL 20% PFA)
When filtering remember the HA backing filter – wet it first, then place 0.2um
polycarbonate filter, shiny side up, on top. Filter as described below.
AAP: after 1 hour fixation, filter 10 mL on one black filter. Place in a
cryovial. Store in -80. Do not rinse.
Label with the next sequential number in the MO notebook and complete the
notebook.
Direct Counts: refrigerate until next morning. Filter 10 mL on one black
filter. Release vacuum. Do not rinse. Close tower, and stain for 3 min with 1 mL
of DAPI staining solution. Filter. Mount on labeled slide with a 25 x 25 mm cover
slip and ~8 uL of oil per filter. The label is the number taken from the direct count
log. Store in slide box at -20 C.
FISH: Refrigerate until next morning. Filter 10 mL on white filters – do 3
filters. Place in 7 mL scintillation vial, Store at -20. Rinse three times with Milli-Q
water dispensed through a 0.2 um filter. Remove PC filter while pump is still
turned on. Label with the next sequential number in the MO notebook and
complete the notebook.
Flow: Fix 1.6 mL seawater with 180 uL of 20% PFA in cryovial in
duplicate for each sample. Store at -80 C. Label with the next sequential
number in the MO notebook and complete the notebook.
DNA/RNA samples:
Pre-filter 1500 mL through 47 mm 0.8 um polycarbonate filter (yellow
plastic set-up). Change the pre-filter a couple of times (will speed things up).
Filter 500 mL per sample on Suropore filter, three times for both whole
water and <0.8 um fraction. (E.g. put 250 mL through for whole water, but put two
filters in one vial). Place in microfuge with 1 mL CTAB.
Final product. 6 tubes (3 whole, 3 pre-filtered) per station.
Label with the next sequential number in the MO notebook and complete the
notebook. Store at -80.
MO June 2011
Chlorophyll:
Filter 100 mL (or until you can see the color) through a GF/F; place in 20
mL scintillation vial. 3 replicates, one filter in each vial. Keep at -20 C.
Nutrients:
Rinse 47 mm GF/F filters three times with 50 mL of seawater, discard
seawater. Filter 150 mL to rinse 125 mL polycarbonate bottle three times, then
filter enough seawater to fill bottle half way. Save filters for DOC. Label and
record in notebook, and freeze at -80 C.
DOC:
For each sample, filter 100 mL to rinse small glass side-arm flask. Pour
50 mL into flask, and add 50 uL concentrated HCl using glass pipette tip. Aliquot
10 mL of mixture into glass vials, 3 replicates. Flame opening of glass vials and
seal. Label and record in notebook and freeze at -80 C.
HPLC pigments:
Filter 400-600 mL seawater onto 25 mm GF/F filter. Fold filter like a taco
and wrap in foil. Place foil with filter in a cryovial. Do 6 filters per sample. Label
and record in notebook, and freeze at -80 C.
Production:
All in centrifuge tubes.
Live – 3 reps
Killed – 1 rep
Leucine addition:
Ten-fold dilution (15 uL plus 135 uL water)
Add 16.2 of diluted leucine to each bottle (use spreadsheet to calc)
See the production protocol over rad bench.
Micro-FISH:
Dark – 15 mL
Killed – 5 mL
KILL the Killed control with 1.5 mL PFA before adding compound (preferably
about 5 min before)
Leucine add (straight from stock, not diluted)
MO June 2011
20 nM: add 16.2 uL of stock solution to live, 5.4 uL to killed
Incubate 1 hour. Kill with 1.5 mL PFA for 1 hour.
Filter 5 mL per White (black for AAP) 0.2 um polycarbonate filter (use wet HA
backing filter). Rinse 2-3 times with milli-Q. Do 3 filters per sample, store all 3 in
one 7 mL scintillation vial at -80 C.
MO June 2011