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 !
