Gambierdiscus Whole Cell Hybridization
Prepare Prehybridization/ Hybridization Buffer.
The Promega filtration manifold has a 14-sample capacity.
The following recipe prepares buffer for 15 samples.
In a 50 mL centrifuge tube labeled “prehybridization” buffer add: 20.4 mL Milli-Q water 6.0 mL 25X SET 300 μL 10% IGEPAL CA-630 300 μL Poly A 10 mg/mL (3-10 freezer in Styrofoam box) 3.0 mL Formamide* (in flammable refrigerator/freezer in 3-30). 
Prepare hybridization buffer: Probe working stock concentration = 200 ng/μL.
For each sample, use 1 mL buffer + 10 μL working stock probe.
Thus, for 14 samples, transfer 14 mL buffer into a 15 mL tube labeled “hybridization buffer and add 140 µL probe (14 x 10 μL).
Prepare 0.2X SET Wash For 15 samples (1 mL per sample): 120 μL 25X SET 14.880 mL Milli-Q water 20ul of Calcifluor (3-30 fridge). 
Place Whatman Cyclopore filter (5 μm, 25 mm), shiny-side up, on the filter unit base with minimal vacuum applied (2.5” Hg = 65 mm Hg).
With continued vacuum, wet filter with Milli-Q, add the o-ring and the chimney.
Tighten by only turning base of the filter chimney!
Discard blue backing filter.
Label the towers with the appropriate sample information.
Include: Site ID and number, Sampling Month and Hybridization Date.
(Use a sticky label that can later be placed onto a microscope slide).
Mix sample well (inverting tube ~6 times) and remove an aliquot of sample and place onto the membrane (record volume to sample used).
Filter each tower to near dryness.
EMPTY CONTENTS OF FILTRATION MANIFOLD (THE “PIG”) INTO METHANOL WASTE CARBOY!
Add 1 mL prehybridization buffer to each tower.
Prehybridize the cells for 5 minutes at room temperature.
Filter each sample to near dryness.
Add 1 mL hybridization buffer containing the oligonucleotide probe.
Cap the tubes and place the filter manifold into a large black plastic bag containing a wet paper towel to help minimize evaporation.
Fold over the bag and seal it with a binder clip.
Place the filter manifold and the tube of 0.2X SET into the incubator and allow the samples to hybridize for an hour at the probe's hybridization temperature.
After incubation, filter each sample to near dryness.
Add 1.0 mL 0.2X SET (50˚C) to each sample (wash step) and incubate for 5 minutes at room temperature.
Filter each sample to near dryness.
While the vacuum is on, remove the chimney (loosen by only turning base of filter chimney!).
Remove the filter from the fritted base and place it on a microscope slide using forceps (minimize the amount of filter surface area that forceps come into contact with).
Add 25-30 μL glycerol/SET solution in equal drops to the filter and mount with a cover slip.
Add the label to the slide.
Store prepared slides cold and dark.
View filters on a fluorescence microscope with the appropriate filter set.
Counts should be completed within 1-2 days of staining.
EMPTY CONTENTS OF FILTRATION MANIFOLD (THE “PIG”) INTO FORMALIN WASTE CARBOY!
