PICODIVa European FP5 program
	Monitoring the diversity of photosynthetic picoplankton in marine waters

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Quick Access
		Download
Sample codes
Sampling protocol	Monthly sampling protocol at coastal stations	Excel file
		Step by step Powerpoint presentation by Fabrice at Barcelona meeting
Measurement protocols	Measurement assignement to each group	Excel file
	Lugol stock solution
	Flow cytometry protocol	Marie et al. 1999 PDF file Presentation by D. Marie (Power point)
	Heterotrophic flagellates stained with DAPI
	Electron microscopy
	HPLC for pigments (including sampling)
	Nucleic acid extraction (Massana)
	DGGE protocol	Presentation by R. Massana (Power point)
	TSA-FISH protocol (including sampling)	Presentation by F. Not (Power point) List of probes to be applied (Excel file)
	Clone library
Supplementary protocols	Electron microscopy (Laure Guillou)
	Nucleic acid extraction
	Dave Scanlan Comparison of different protocols done during MIDAS workshop
	FISH from Chris Scholin
	SSCP C.Tebbe protocol (Power point)

Sampling Protocol for coastal stations

Sampling codes

Coastal stations	Cruises
ST_YYMMDD_Parameter	CRU_CTD_BOT_Parameter
ST     RA Roscoff Astan   RM Roscoff Morlaix Bay   HE Helgoland   BL Blanès Bay   AQ Eilat Gulf of Aquaba	CRU     PRO PROSOPE   MAT99 MATER99   HIV00 HIVERN00

Exemples:

• HE_000420_FISH_Euk for the FISH eukaryotic filter taken at the Helgoland Station on the 20 April 2000
• PRO_35_10_FCM for the sample taken for flow cytoemetry in bottle 10 of CTD 35 during the PROSOPE cruise.

Finalized set of parameters for monthly survey of year 2

Download table as Excel file

Parameter	Method	Sample prefiltration	Replicates	Sample volume (ml)	Sample processing	Sample Preservation	Sample analysis	Sample analysis done by	Remark
Temperature								Locally
Salinity								Locally
O2								Locally
Light (Secchi)								Locally
NO3-						-20 °C		Locally
NO2						-20 °C		Locally
NH3								Locally
PO4						-20 °C		Locally
Si(OH)4						-20 °C		Locally
Chlorophyll a	Fluorometry					-20 °C		Locally
Phytoplankton composition	Microscopy		2	50	Lugol 0.25%	Store in the dark at RT	Sedimentation and observation with inverted microscope (volume to sediment : 20-100mL)	Locally
Picoplankton taxonomy	TEM of whole mounts	< 3 um	3	200	1 fixed with osmic acid 1 fixed with 0.25 % glutaraldehyde and 1% acidic Lugol. 1 fixed with 1% glutaraldehyde in 0.2 M cacodylat. See Wenche protocol for details on fixation	Store in the dark at 4° C	EM protocol by Wenche	Wenche	Samples should not be accumulated but sent immediately to Wenche.
Heterotrophic eukaryotes abundance	Epifluorescence micorscopy	Total		90	Fix 90 ml of seawater with 10 ml ice-cold glutaraldehyde 10% (1% final) then filter (see protocol by Ramon)	as filters at -20 °C	See protocol by Ramon	Locally
Picoplankton abundance (autotrophs)	Flow cytometry	Total		1.5	1% paraformaldehyde+ 0.1 % glutar	in cryovials at - 80°C	Unstained on flow cytometer	Locally	See Marie et al 99
		<3 um		1.5	1% paraformaldehyde+ 0.1 % glutar	in cryovials at - 80°C	Unstained on flow cytometer	Locally	See Marie et al 99
Bacteria abundance	Flow cytometry	Total		1.5	1% paraformaldehyde+ 0.1 % glutar	in cryovials at - 80°C	SYBR Green stained	Locally	See Marie et al 99
Pigments	HPLC	Total		1000	Filter on 25 mm	as filters at -80°C	HPLC protocol	Mikel	See Mikel protocol
		< 3 um		1000	Filter on 25 mm
		> 3 um		1000	Filter on 47 mm
FISH for eukaryotes	FISH-TSA	< 3 um	3 filters	200	See detailed protocol by Fabrice. It is critical that 1 - fixation with PF does not exceed one hour and 2 - filters be always kept humid during filtration and dehydration	as filters at -80°C in individual plastic box	FISH Protocol	Fabrice	See FISH-TSA protocol
FISH for prokaryotes	FISH-TSA	< 3 um	2 tubes	50	glutaraldehyde (0.1% v/v) final concentration for 10 min at 4 oC	as 50 mL flacon tubes at -20°C	FISH Protocol	Dave
Picoplankton diversity	DGGE eucaryotes	< 3 um		5000	Sample collected on Sterivex 0.2 um Millipore unit (see Rmaon protocol)	as Sterivex filters at -80°C	DGGE protocol	Ramon	A single DNA sample will be collected. After DNA extraction, the DNA will be split between the different groups
	DGGE procaryotes						DGGE protocol	Dave
	Quantitative PCR						Fluorescent PCR	Isabelle Roscoff
	DNA chips						DNA chips	Klaus
Remark				This is the volume for a single sample		When mentionned -80°C samples must be stored in a deep freezer, not a regular freezer		All frozen samples are to be sent on dry ice

Prefiltration on 3 um

For all measurements on picoplankton population, samples will need to be pre-filtered through 3 um. The best way will be to use in line fitler filter (25 or 47 mm) with a peristaltic pump at low rate. This will ensure sample homegeneity

Filter used: 47 mm polycarbonate 3 um
Volume filtered:

• 6 L for FISH, DGGE, DNA hybridization and flow cytometry
• 2 or 5 L for HPLC depending whether 25 or 47 mm filters are used
  

Protocol for clone libraries on 3 um filtered samples

No fixed protocol will be used for library building (in particular choice of DNA extraction, , in order to maximize the diversity of recovered sequences. However we agreed on a set of primers and enzymes for screening, as well as on some recommendations.  See the library protocol below.

Parameter	Sample Volume (ml)	Sample storage	Sampling protocol	Analysis protocol
Clone library	5000	-80°C	R. Massana protocol (recommended) D. Scanlan protocol	Library building and analysis

Protocol for cultures on 3 um filtered samples

Each starting culture will be duplicated. Starting cultures are done in 50 ml Falcon flasks.

Parameters to be varied (not all combinations though):

Purification methods

Recommended Protocols

Lugol stock solution (recipe from : Phytoplankton manual, Unesco, Sournia 1978) Dissolve 100g of KI in 1 L of distilled water Add 50g of iodine (crystalline) Add 100 mL of glacial acetic acid. Decant to remove precipitates

Counting pico and nannoplankton by epifluorescence microscopy (Ramon) Fixation - Fix 90 ml of seawater with 10 ml ice-cold glutaraldehyde 10% (1% final concentration) - Store samples at 4°C until processing the samples (before 24 hours) Preparation of filters - Place a 0.8 µm (25 mm ø, cellulose acetate) filter on the base of the filtration system and wet it with sterile water. Put on top of it a 0.6 µm (25 mm ø, polycarbonate) filter. - Add 20 ml of fixed seawater on the tower (the volume depends on the abundance; check it and adjust to convenience). - Filtrate the sample until the seawater volume is reduced to 5 ml. - Add 50 µl of DAPI working solution (5 µg/ml, final concentration) and stain for 5-10 min. - Put one drop of low fluorescent oil on one slide (Cargille, Nikon), place the filter on the drop and wait until the filter became transparent. - Add another oil drop on top of the filter and put the cover slide. - Keep the slides in special plastic boxes frozen at -20°C Counting flagellates on the epifluorescence microscope - Observe the slides with UV excitation: pico and nanoflagellates appear as round cells with nucleus (a brighter area). Sometimes the flagella is also visualized. - Immediately, change to blue light (485nm excitation, 530 nm emission wave-length, and 505 nm dichroic mirror) to discriminate between colorless cells from cells with plasts and pigments. This makes the possibility to separate heterotrophic from phototrophic nanoflagellates. - Counts of each group of nanoflagellates (phototrophic and heterotrophic) are made separately, enumerating the nanoflagellates through transects (5 - 10 mm). For each sample is recommended to count at least 50-100 cells. Hence, we will make the necessary number of transects (3 transects of 5 or 10 mm are enough). Dye working solution (DAPI) - Add 1 ml of filtered seawater (0.2 µm) to the 10 mg DAPI vial. - Add the 10 mg/ml of DAPI to 19 ml of sterile filtered seawater (0.2 µm). We will have a concentration of 0.5 mg/ml de DAPI. - Filter this solution through 0.2 µm (Swinex and sterile syringes), and put it in a clean 20 ml vial. THIS IS THE WORKING DAPI SOLUTION - Take 20 criovials and add to each one1 ml of DAPI working solution. Keep the criovials frozen (-20°C) until its use.

Electron microscopy for natural samples (Wenche Eikrem) New procedure for samples (as of May 2001) ==> Use dark glass bottles if possible Bottle 1: Osmium fixation Total volume ca 200 ml To 200 ml of prefiltered (3 microns) sample: add 10 drops of 2% osmiumtetroxide. Turn the bottle gently a couple of times. Bottle 2: 1% glutaraldehyde in 0.2 M cacodylat (final concentrations) Total volume ca 400ml To 200 ml of 0.4 M cacodylat: add 16 ml of 25% (or 8 ml of 50%) glutaraldehyde (mix), add 200 ml of prefiltered (3 microns) sample. Turn the bottle gently a couple of times. Stock solution of 0.4 M cacodylat: Dissolve 85, 6 g of cacodylat in 1000 ml of distilled water. Adjust pH to ca 7.8. Bottle 3: 0.25 % glutaraldehyde and 1% acidic Lugol (final concentrations) Total volume ca 200 ml To 2 ml of 25% (or 1 ml of 50 % ) glutaraldehyde and 2 ml of acidic Lugol: add 200 ml of prefiltered (3microns) sample. Turn the bottle gently a couple of times. Stock solution of Lugol, 10g of KI, 5g of I2 and 10ml of glacial acetic acid in 100 ml of distilled water.

HPLC analysis of pigments (Mikel Latasa, Barcelona) Note: For PICODIV all filtered volumes are set to 1000 mL (see big table above) Water collection from field samples. Sample a minimum of 2 L of water for 25 mm filters and 5 L for 47 mm filters. In case that on board filtering is impossible, place the bottle in a dark bag and at 4 °C or less. Try to filter the sample within 2-3 h maximum from its sampling.. Filtration procedures. Always use 200 mm or less vacuum/pressure. 25 mm filters are strongly recommended, although 47 mm filters are fine if more water is available. It is OK to use always 47 mm polycarbonate filters. With 47 mm filters more acetone is needed to extract the pigments = sample is more diluted = the chromatographic peaks are smaller and it is more difficulties to identify them. Filter as much water as possible (2 L and 5 L for 25 and 47 mm filters, respectively). Modify the volumes according to the phytoplankton concentration. A very good amount of pigment for subsequent peak identification is about 2 microgram (ug) of chlorophyll per 25 mm filter. Below 0.5 ug per filter correct peak identification becomes harder (it depends on the equipment). If there is not a clue about pigment concentration in the field, use filtering time as a criteria: the maximum filtering time should not exceed much more than 1 hour. Keep a dim light while filtering. Freeze the filters immediately at -80 °C or, better, in liquid nitrogen (-20 °C freezing may alter the pigments). There are two alternative filtering systems: vacuum filter through 3 um 47 mm polycarbonate filter. Collect the filtrate and refilter through GF/F filter. pressure filter through sequential 3 um polycarbonate and GF/F filters. Pigment extraction Place the filters in closed polypropylene tubes with 3 mL 90% acetone (do not use methanol). GFF filters should be folded and squeezed while wrapped in absorbing paper to remove all the water content. Store the extracts at 4 °C for 12 h or more (we have seen that there is not degradation of pigments at least during the first 48 h). After this time, destroy the filter with a Teflon grinder or in a mill with glass beads avoiding to heat the extract (an ice bath is usually enough). Centrifuge for 15 minutes at 4 °C. Transfer the supernatant fluid to another vial. Store it at -20 °C until analysis (we have seen that there is not degradation of pigments, at least during the first 48 h). HPLC procedure This specific protocol is a modified version of Zapata et al.'s method (2000) and significantly improves pigment separation from previous protocols. The column is a C8 Waters Symmetry (150 x 4.6 mm, 3.5 µm particle-size) protected with a 3 x 4.6 mm guard column containing the same stationary phase. The column is thermostated at 25 °C employing a water jacket (Alltech) connected to a recirculating water bath. The temperature control is extremely important for this kind of column. 100 to150 uL of a mixture of 1 mL of extract and 0.2 mL H2O are injected onto the chromatographic system and run according to the following specifications. Eluent A = methanol:acetonitrile:0.25M pyridine aqueous solution adjusted at pH 5.0 with acetic acid (50:25:25 v/v/v) and Eluent B = acetonitrile:acetone (80:20 v/v). Elution gradient is as follows (percentages are complemented with solvent B): 0' 100%A, 10' 77%A, 23' 77%A, 25' 65%A, 35' 60%A, 38' 25%A, 46' 15%A, 48' 0%A, 57' 0%A, (we are trying to shorten the running time without loosing resolution). The flow rate is 1 mL min-1. The absorbance signal is recorded at 440 nm for routine samples or with 400-700 nm diode-array spectra for specific samples. Pigments are quantified with response factors obtained from commercial standards (The International Agency for 14C Determination, former Water Quality Institute, Denmark)

Remarks from Mikel based on the analysis from the first Roscoff samples (May 2000) Use 25 mm diameter filters. With 47 mm filters we need to use more acetone to extract the pigments = we dilute the sample = we see less peaks and we have more difficulties to identify the ones we actually see. I know the Nuclepore filter cloggs faster. In this case it's OK to use 47 mm because we still can extract these filters in a small volume of acetone. Filter more volume of sample. One liter is more than enough when there is a lot of material (which wasn't the case). I would go for the 2 liters as a rule, and modify the volumes according to the phytoplankton concentration. A very good amount of pigment for subsequent peak identification is about 2 microgram of chlorophyll per 25 mm filter. Below 0.5 ug per filter we can only try our best (it depends on the equipment). If the time or the water is tight at the sampling site, at least try to collect as much as possible for the non-fractionated sample (to help peak identification, which needs higher concentrations than peak quantification).

Nucleic acid extraction from seawater samples (Ramon Massana) Collecting biomass for marine DNA extraction: Collect 5 liters of surface seawater Filter sample with a peristaltic pump, having two filters in sequence,first a 47 mm polycarbonate filter of 3 µm poresize, and then a Sterivex unit (Millipore) of 0.2 µm pore size. Cover both filters with lysis buffer (40 mM EDTA, 50 mM Tris-HCl and 0.75 M sucrose) and store at -80°C. Nucleic acid extraction Add lysozyme (1 mg ml-1) to the Sterivex filter and incubate at 37°C for 30 min. Add proteinase K (0.5 mg ml-1) and SDS (1%) to the filter and incubate at 55°C for 2 hrs. Extract the lysate twice with phenol: chloroform: isoamyl alcohol (25:24:1;pH=8) and once with chloroform: isoamyl alcohol (24:1). Wash and concentrate the aqueous phase in a microconcentrater (Centricon 100, Millipore) and obtain a final volume of DNA extract of 200 µl. Quantify DNA extract by a Hoescht dye fluorescence assay and check the integrity by agarose gel electrophoresis. Store extracts at -70°C until analysis.

DGGE (Massana and Diez) PCR Use 1 ng of whole microbial DNA as template for Polymerase Chain Reaction (PCR) amplification of bacterial or eukaryal SSU rDNA. The reactions contain 200 µM of each of the deoxynucleoside triphosphates, 0.3 µM of each of the primers, 1.5 mM MgCl2, 1x PCR-buffer and 1 Unit of Taq DNA Polymerase in a final volume of 50 µl. Primers Eukarya: Eukaryal specific forward primer EukA (Medlin et al. 1988) and universal reverse primer 516r (ACC AGA CTT GCC CTC C) with a 40 bp GC-clamp, which amplify a 560 bp DNA fragment of eukaryal 18S rDNA. PCR program Initial denaturation at 94°C for 5 min; 10 touchdown cycles of denaturation (at 94°C for 1 min), annealing (at 65 to 55°C for 1 min, decreasing 1°C each cycle) and extension (at 72°C for 3 min); 20 standard cycles of denaturation (at 94°C for 1 min), annealing (at 55°C for 1 min) and extension (at 72°C for 3 min), and a final extension at 72°C for 5 min. PCR product check Four µl of the PCR product are verified by electrophoresis on a 0.8% agarose gel stained with ethidium bromide, and the DNA yield of the PCR is quantified loading a standard in the same gel (Low DNA Mass Ladder, GIBCO BRL). DGGE Denaturing Gradient Gel Electrophoresis (DGGE) is performed with the DGGE-2000 system (C.B.S. Scientific Company) as described in Muyzer et al. (1997). Gel casting A 6% polyacrylamide gel is casted by mixing two stock solutions of acrylamide (37.5:1 acrylamide: bisacrylamide) containing different amounts of DNA denaturant agents: 40 and 80% for bacterial PCR products and 45 and 65% for eukaryal PCR products (100% denaturant agent is defined as 7 M urea and 40% deionized formamide). The reproducibility of the gradient is obtained by using a two chambers gradient maker and controlling the flow of the acrylamide into the plates with a peristaltic pump at around 5 ml min-1. The gradient is overlaid with nondenaturant acrylamide in order to obtain well polymerized slots. The gel is casted several hours before loading. Gel running 800 ng of PCR product (typically 40 µl of the PCR product) are loaded in the gel slots with a Hamilton syringe for each sample (a maximum of 18 samples per gel). The gel is run at 100 V and 60°C for 16 h in 1x TAE buffer (40 mM Tris base [pH 7.4], 20 mM sodium acetate, 1 mM EDTA). Gel staining We used the DNA stains GelStar (FMC BioProducts) or SYBRGold (Molecular Probes). One of the glass plates is detached from the gel and 15 ml of the stain solution (15 ml of 1x TAE with 3 µl of the stain stock solution) are added covering the whole gel. The gel is stained for 30 minutes in the dark, rinsed with a large volume (around 500 ml) of 1x TAE buffer, removed from the glassplate and transferred carefully to a UV transparent gel scoop (Sigma). The gel is visualized with U.V. in the Fluor-S MultiImager (Bio-Rad) with the Multi-Analyst software (Bio-Rad). High-resolution images (1312 x 1034 pixels, 12-bits dynamic range) are saved as computer files (4.6 Mb). Quantitative analysis of DGGE fingerprints The computer image of the gel is analyzed with the Diversity Database software (Bio-Rad) as explained in Schauer et al. (2000). The software performs a light intensity profile through each lane, detects the bands, and calculates the relative contribution of each band to the total band signal in the lane after applying a detailed rolling disk as background subtraction. Intensity values are recalculated for all the bands of one lane to sum up to 100% relative intensity. The different lanes from the same gel are compared and the bands occupying the same position in the gel are identified. A matrix is constructed for all lanes, taking into account the presence or absence of individual bands, and the relative contribution of the band to the total intensity of the lane. This matrix was used to calculate a distance matrix using Euclidean distances (Systat 5.2.1). A dendrogram relating all samples is obtained with the unweighted pair group average linkage method (UPGMA) in cluster analysis (Systat). The number of bands and the intensity of each band can be used to calculate the Shannon diversity index (H') with the following formulae: where n is the number of bands in the sample and pi the relative intensity of the ith band.

TSA-FISH protocol for eucaryotes (F. Not) (from Amann 1995 and modified from Schönhuber et al. 1997, Adapted by Biegala and Simon for bacteria. Updated nov 00 for eukaryotes) A. Cell preparation A1. Fix the cells Add 1 vol. of ice cold 10% paraformaldehyde (made fresh with 1X PBS, aliquoted and stored at -20°C) to 9 vol. of cell culture (1% final PFA concentration). Incubate 1h at 4°C in dark. Do not exceed this 1h duration or there will be heavy cell loss.  A2. Filtration Use clean filtration devices (Tulip and support washed with 1% HCl and sterilized if necessary). Filtered cell sample on (25 mm, 0.2 µm pore size) inorganic membrane filter (Anodisc, Whatman, Maidstone, UK), with a 10 mHg pump. Add 2 ml of 50% EtOH, leave 3 min, and filter. Add 2 ml of 80% EtOH, leave 3 min, and filter. Add 2 ml of 100% EtOH, leave 3 min, and filter. The filters must never dry out (this is a critical point) A3. Storage and alteration of cell wall In cleaned plastic box at -80°C for several months (3 months tested) B. In situ hybridization B1. Hybridization The stringency of the hybridization must be empirically optimized (range between 37-55°C) higher stringency can be achieved by the addition of formamide to the hybridization buffer. Hybridization is done in a sealed moisture chamber to prevent evaporation of the hybridization solution, which can result in non-specific probe binding to the cell. Soak a piece of Whatman 3MM paper (2.6 cm/6 cm) with 0.8 ml of hybridization buffer (0.01% SDS, 40% Formamide for eukaryote probes) and intraduce in humid chamber (50 ml polypropylene screw top tube, FISHER). Allow to equilibrate several minutes at 35°C. Put 10 µl of hybridization buffer slide wells. Add 1µl of oligonucleotide probe HRP labelled and 1µl of non HRP labelled competitor probes when necessary (probes concentration: approx. 50 ng/µl). Cover with filter fragment (cells on top, they should not be in contact with the buffer, to avoid swimming around) Quickly transfer the slide in the pre-warmed humid chamber. Incubate 2h to 3h at 35°C. B2. Wash Immerse the filter (cells on top) in plastic wells in 5ml wash buffer (0.01% SDS with a concentration of NaCl which correspond to the formamide stringency during hybridization see correspondence table below) Cover with plastic lid and incubate in dark, 2 times 20 min at 37°C. B3. Rinse briefly Immerse the filter in plastic wells in 5 ml ddH2O at room temperature B4. Equilibrate Immerse the filter (cells on top) in plastic wells in 5ml TNT buffer Cover with plastic lid and incubate 15 min room temperature, in dark B5. TSA reaction (Tyramide Signal Amplification) Thaw an aliquot of dextran sulfate (Sigma) 40% Mix 1:1 with amplification diluent (Kit: NEN Life Science Products) Mix 1:50 fluorescein tyramide or tetramethylrhodamine tyramide (Kit: NEN Life Science Products) with the mixture of dextran sulfate and amplification diluent Add 10µl of the fluorochrome tyramide on the filter (cells on top) Incubate at room temperature in dark for 30 min B6. Wash Immerse the filter (cells on top) in plastic wells in 5ml TNT buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05 % TWEEN 20) Incubate 2 times 20 min at 55°C B7. Rinse briefly Immerse the filter in plastic wells in 5 ml ddH2O at room temperature. Allow the filter to dry at 37°C until complete dehydration. At this step either repeat a second hybridization with the other fluorochrome or follow the protocol. B6. DAPI labelling Put 10µl of DAPI <5µg/ml on coated slide on filter, cells on top Incubate for 10 min at room temperature in the dark B7. Wash Immerse the filter (cells on top) in plastic wells in 5ml double distilled H2O Incubate 10 min at room temperature in dark Allow the filter to dry on slide at 37°C B8. Mount slide for epifluorescence observations Cover filter (cells on top) with 20 µl of antifading Citifluor agent. Cover with cover slip and seal with varnish The slides are then kept at 4°C in dark and can be further analyzed and observed within two weeks.   Stock solutions for FISH of eukaryotes 10% paraformaldehyde fixative (PFA10) **MADE FRESHLY**Stored at -20°C then at 4°C For a total volume of 100 ml. Add 65 ml of ddH2O heated at 60°C Add 10 g of paraformaldehyde Add 1 drop of 2M NaOH and stir rapidly until solution has nearly clarified (1-2 min). Remove from heat Add 33 ml of 3X PBS (pH 7.2) Check pH of entire solution should be 7.2 if not adjust with concentrated HCl Filter through 0.2 µm filter. Quickly cool at 4°C and store at 4°C. Hybridization buffer (0.01% SDS, 50% to 36% Formamide according to probes) Deionised formamide (Sambrook et al. 1987) In a beaker 15 ml of formamide Add a spoon of resin Homogenize for 1h (the blue bids become orange) Filter the formamide on Whatman paper (the Whatman absorb 2 ml of fluid!) 50% to 36% formamide 0.9 M NaCl 20 mM Tris pH 7.5 0.01% SDS For 10 ml final: (aliquoted and kept at -20°C) ddH20 3.6 ml formamide (36%) to 5 ml formamide (50%) 1.8 ml NaCl 5M 200 µl Tris 1M pH 7.5 100 µl 1% SDS 2 ml Blocking buffer 10% (Blocking Reagent : Boehringer Mannheim) Filter the mixture with seringe & filter Aliquots of 1 ml Wash buffer Note: it should have the same Tm as the hybridization buffer even though there is no formamide and the equivalent stringent power is created by increasing NaCl concentration (see corresponding tables). NaCl eq. 50%, 40% Formamide 5 mM EDTA 0.01%SDS Tris 20mM pH 7.5 For 50 ml of wash buffer: 180 µl (eq 50%F) 460 µ (eq 40%F) of NaCl 5 M 500 µl EDTA 0.5 M 500 µl 1% SDS 1 ml Tris 1 M pH 7.5 % Formamide [NaCl] in mol/l ml of NaCl for 50 ml final 0 0.900 9.000 5 0.636 6.300 10 0.450 4.500 15 0.318 3.180 20 0.225 2.150 25 0.159 1.490 30 0.112 1.020 35 0.080 0.700 40 0.056 0.460 45 0.040 0.300 50 0.028 0.180 TNT buffer Tris 0.1M pH 7.5 NaCl 0.15M Tween 20 For 250ml 25ml Tris 1M pH 7.5 7.5ml NaCl 5M 0.185ml Tween 20 CitiFluor (AF3 mountant solution, Univ. Of Canterbury) Stock solution aliquoted and kept at 4°C. Working solution = unchanged work solution.

Clone library contruction and analysis (K. Valentin) Taking samples Collect ca. 3-5 L surface water Filter asap through a 45 mm 3m m filter at 200- 400 mbar. Filter the filtrate through a 45 m m 0.45 or 0.22 m m Filter (one filter will hold 200 - 1000 ml of prefiltered seawater) at 200-400 mbar vacuum. IMMEDIATELY soak the filter in 20 m l TE (= 10 mM Tris, 100 mM EDTA pH 7.5-8) and transfer to an Eppendroff cup. IMMEDIATELY shock-freeze the cup in liquid Nitrogen, store at –20° C for weeks or at -80° C for months or years. (note: EDTA and shock-freezing will inhibit DNAses) Isolating DNA Recommended method is CTAB. PCR Reaction mix: 100 ng DNA, 2 u TAQ polymerase (Perkin Elmer), 1F and 1528R primers, 1,5 mm MgCl2, no BSA, MWG nucleotides in a volume of 50 m l. Cycles: 5 min 94° C (2 min 94° C, 2 min 53° C or 50° C, 4 min 72° C)x35-40, 5min 72° C, 4° C. For each sample two reactions at annealing temperatures of 50° and 53° C were carried out and combined. Annealing temperatures below 50° C resulted in multiple products of the wrong size. Table 1: Primers and enzymes for clone libraries Name E. coli Tm Sequence (5' to 3') Ref Remark PCR primers Euk Medlin Medlin et al. 1988 The lower case bases correspond to restriction sites Euk1f 1 50 or 53 ccgaattcgtcgacAACCTGGTTGATCCTGCCAGTA Euk1528r 1528 50 or 53 cccgggatccaagcttGATCCTTCTGCAGGTTCACCTAC PCR primers Euk Moon    Moon et al. 2000   Euk328 2 55 ACCTGGTTGATCCTGCCAG   Euk329r 1528 55 TGATCCTTCYGCAGGTTCAC   An comparative analysis of the specificity of 18S primers from Medlin and Moon has been perfomed by Rupert de Wachter and his group (Antwerpen University).  SY Moon primers are more general than those of Medlin - download document (please treat these data as unpublished private communication) PCR primers Photo Scanlan      For 16S cyanos/plastids ProkPhoto106f 106   ProkPhoto1313r 1313       Screening primers           Euk528f 528   ccg cgg taa ttc cag ctc     Screening enzymes    HaeIII      Sequencing primers Tm based on G/C Medlin *82F GTG AAA CTG GAA TGG CTC AT *528F 58 GCG GTA ATT CCA GCT CCA A 690F 54 GIS AGA GGT GAA ATI CTY RGA *1055F 61 GGT GGT GCA TGG CCG TTC TT *1400R ACG GGC GGT GTG TRC AAA GG *1055R 72 ACG GCC ATG CAC CAC CAC CCA T 690R 62 ATC CAA GAA TTT CAC CTC TGA C *536R 64 AAT TAC CGC GGC KGC TGG CA * = the ones we use regularly reading 800 bp with a Licor sequencer, get 50/100 bp overlap between the primers) These sequencing primer are slightly modified form the original ones published by Elwood et al. 1985 because they had to be modified for length and GC content for the Licor sequencer. They all give good sequence. Unfortunately I used the original version of the 528f to make the sequencing primer form the clone libraries. You will note that the sequence is slightly shifted from the Licor primer. Purifying PCR products Products (500 ng) were electrophoresed on an agarose gel (1%) in the presence of Crystal Violet (add 0.5 - 1m l of a 2 mg/ml stock soln. Per ml gel) without Ethidium bromide. This allows visualisation of the DNA band under normal light. Bands were cut out and purified from the agarose with easypure" from Biozym. Cloning of PCR products Purified PCR products were cloned using the TOPO XL zero background kit from Invitrogen. With this kit a standard reaction produces 500 - 1000 clones with an efficiency >95%. Therefore it is possible to generate more than 10,000 - 20,000 18S clones from 500 ml filtered seawater. Selection of clones Minipreps (Qiagen) carried out from randomly picked clones Screening is done with HaeIII Unique clones are sequenced with primer Euk528f : this primer binds next to the most variable region of the 18S molecule, i.e. the V4 region Analysis of clones All selected clones will be sequenced in each direction with primers to be defined by L. Medlin

Other useful protocols

Electron microscopy for natural samples (Laure Guillou)

• Fix 9 ml of sample with 1 ml of fixing solution (Laure??)
• Let sit for at least 1h and up to 2-3 h
• Write down fixation time
• Put 1.5 ml in 6 2 ml Ependorf each containg a small 0.5 mL Ependorf with a formvar coated grid at the bottom
• Centrifuge 10 min à 6 000 rpm
• Eliminate supernatant up to level of second inside small tube
• Centrifuge a second time 5 min à 6 000 rpm
• Remove all supernatant and get the small tube out with a needle
• Take out the grid and rince 2-3 times by immersion in DW
• Store grid in grid box

Nucleic acid extraction from seawater samples (Dave Scanlan)

Filter 4 - 10 litres of seawater onto a 0.2 um pore size Gelman Supor-200 47 mm diameter filter on a vacuum of approx. 5-8 in Hg. Use of a 0.45 um pore size Gelman Supor-450 47mm diameter filter improves rate of filtration significantly and is fine for both Synechococcus and Prochlorococcus. The Supor filters are advantageous because they have:

1. low level of contaminants;
2. high filtration rates;
3. dissolve entirely in phenol during the extraction process.

Protocol I: Mainly as set out in Kerkoff and Ward (1993). AEM 59:1303-1309

• Distribute 0.5 ml of 0.5 M EDTA over filter. Store filter at -70 C in a petri dish.
• Roll filter into a 10 ml liquid tight sterile test tube (resistant to phenol and chloroform) so they uncurl on the sides of the tube.
• Add 600 ul of cold 50 mM glucose-10 mM EDTA-25 mM Tris pH8.0 (GTE buffer), 8 ul of 250 mg/ml lysozyme, and 200 ul 0.5 M EDTA pH8.0.
• Incubate on side (so that filter is immersed) at RT for 10 mins rolling tube occasionally.
• Lyse cells with 100 ul of 10% SDS and vortex well.
• Extract immediately in 1.5 ml of phenol-chloroform-isoamyl alcohol (25:24:1). Such a high volume of phenol/chloroform is required for complete dissolution of the filters into the organic phase. Vortex well. Aliquot into eppendorf tubes.
• Centrifuge on maximum for 5 min at RT. Transfer aqueous (upper) phase to fresh tubes.
• Extract with chloroform/isoamyl alcohol, and spin as above. Transfer aqueous phase to fresh tubes.
• Precipitate with 1 volume of isopropanol and 0.4 vol of 7.5 M ammonium acetate. Incubate at RT for 10 minutes. [For Red Sea water, we find that precipitation with ethanol causes a massive salt precipitation - so we avoid it.]
• Centrifuge on maximum for 15 min at RT.
• Wash pellets in ice cold 70% ethanol.
• Allow pellets to dry on table top for about 10 min.
• Resuspend in a volume of 50 ul TE2 (10 mM Tris, 0.1 mM EDTA, pH8.0).

  Yield: 1 - 4 ug DNA depending on site and season.

Protocol II: Mainly as set out in Gordan and Giovannoni (1996). AEM: 62(4): 1171-1177.

• Place filter in 5 ml cryovial. Add 3 ml lysis buffer (20 mM EDTA, 400 mM NaCl, 0.75 M sucrose, 50 mM Tris pH9.0) to fully immerse filter. Freeze in liquid nitrogen and store at -70 C. The authors freeze their filters at -20 C as well.
• Thaw sample on ice.
• Add a) 350 ul 10% SDS and mix. b) 33.5 ul 10 mg.ml-1 Proteinase K and mix.
• Incubate at 37 C for 30 min ensuring that filter is fully immersed.
• Incubate at 55 C for 10 min.
• Transfer lysate and filter to centrifuge tube that is resistant to phenol and chloroform. (I use 4 x 2 ml Eppendorfs, cutting the filter into 4 before placing into the 5 ml cryovial above.)
• Extract once in 3 ml phenol:chloroform:isoamyl alcohol (25:24:1)
• Extract once in chloroform:isoamyl alcohol (24:1)
• Precipitate with 1 volume of isopropanol and 0.4 vol of 7.5 M ammonium acetate. Incubate at RT for 10 minutes. [For Red Sea water, we found that precipitation with ethanol causes a massive salt precipitation - so we avoid it.]
• Centrifuge on maximum for 15 min at RT.
• Wash pellets in ice cold 70% ethanol.
• Allow pellets to dry on table top for about 10 min.
• Resuspend in a volume of 50 ul TE2 (10 mM Tris, 0.1 mM EDTA,pH8.0).

Comparison of DNA extraction methods with DGGE from MIDAS workshop

We did this test during MIDAS workshop II in the Alicante solar salterns in May 1999. If you want to see a brief description of the salterns and the workshop you can check the MIDAS web page : http://www.icm.csic.es/bio/projects/midas

What we did was to have four different groups of people use their respective protocols for extraction of DNA in several ponds along the salinity gradient, and then we run a DGGE. The most laborious protocol was Ramon's and the easiest the one from Bergen, where they directly PCR from 1 ml of sample. I enclose the picture for half of the ponds (the other half shows the same). The agreement between methods is striking, while the differences between ponds are substantial.

The lanes are (starting from the left):

1. 11% salinity pond Bergen protocol
2. ---- Alicante protocol
3. ---- Plymouth pprotocol
4. ---- ICM protocol
5. 8% salinity pond Bergen protocol
6. ---- Alicante protocol
7. ---- Plymouth pprotocol
8. ---- ICM protocol
9. 5.4% salinity pond Bergen protocol
10. ---- Alicante protocol
11. ---- Plymouth pprotocol
12. ---- ICM protocol
13. 4% salinity pond Bergen protocol
14. ---- Alicante protocol
15. ---- Plymouth pprotocol
16. ---- ICM protocol

FISH (C. Scholin, modified communicated by Linda Medlin)

Solutions

25X SET buffer:(250 ml)

• 3.75 M NaCl (54.8 g)
• 25 mM EDTA (12.5 ml of 0.5 M stock)
• 0.5 M Tris (15.1 g)
• add 200 ml dH₂O, adjust pH to 7.8, fill up to 250 ml, filter-sterilize using 0.2 µm filter

Saline EtOH fixative (freshly prepared for every experiment!)

• 25 ml Ethanol
• 2 ml dH₂O
• 3 ml 25X SET

Hybridization buffer:

• 189.0 ml dH₂O
• 48.0 ml 25X SET
• 2.4 ml 10% IGEPAL-CA630
• filter-sterilize, then add 0.75 ml poly A (stock 10 mg/ml)

5X SET buffer:(25 ml)

• 5 ml 25 X SET buffer + 20 ml dH₂O, filter-sterilize

Citifluor/DAPI-Mix:

• 1,5 µl DAPI (1 µg/µl)
• 1,0 ml Citifluor
• 0,5 ml dH₂O

 Protocol

• set up filtration unit
• filter sample, use the least amount of vacuum that is possible
• add some ml of freshly prepared saline EtOH, incubate at room temperature for 1 h, filter
• add some ml of hybridization buffer, incubate at room temperature for 5 min. filter
• briefly dry filter on Whatman paper, filters can be stored at 4°C for some time (at least 3 weeks) or processed immediatly
• cut filter into pieces
• put filter piece onto slides, apply 54 µl hybridization buffer with 6 µl probe (50 ng/µl stock) directly onto filter piece
• hybridize slides at hybridization temperature (determine empirically) in a moisture chamber in the dark
• briefly dry filter on Whatman paper, put it back onto clean slide
• apply 100 µl of pre-warmed 5X SET buffer, incubate again in moisture chamber at hybridization temperature for 5 min
• briefly dry filter on Whatman paper, put it back onto clean slide
• apply 30 µl of Citifluor/DAPI mixture directly onto filter, mount cover slip and seal
• view slides using fluorescence microscope

Last updated 29 April 2002