TITLE: Inorganic polyphosphate in microalgae: A DAPI-based quantification in microtiter plate 
AUTHORS: Yingyu Hu, Zoe V Finkel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The DAPI-based fluorometric quantification of polyphosphate in microalgae has been widely used in field samples since the method was published by Martin P. et. al., where fluorescence of DAPI-stained samples is analyzed in quartz cuvettes by spectrofluorometer. In order to minimize the photobleaching of DAPI and reduce the consumption of reagent, time and labor, we have now scaled this method to  96-well black microtiter plate. Regarding to the matrix effects in microplate, the calculation has been modified accordingly. </div><div class = "text-block">Our method permits processing large numbers of samples by using only 250 uL of extracted sample and 30 uL of DAPI (100 uM). A lid with black film can protect all DAPI-stained samples from photobleaching.</div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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[STEPS]
?. [Preparation of reagents]
Proteinase K
?. [Preparation of reagents]
PolyP primary standard stock
?. [Preparation of reagents]
Tris buffer
?. [Preparation of reagents]
In a 1 L volumetric flask, top  Tris buffer to 1 L with MilliQ
20 mL
?. [Preparation of reagents]
Filter through Rapid-flow and store at
0 Room temperature
?. [Preparation of reagents]
Weigh one glass pellet of polyP (45) and write down the weight.
?. [Preparation of reagents]
Dilute to 100 mL with Tris
?. [Preparation of reagents]
Aliquot primary stock into 10~50 uL per microtube with Stepper and store at
-20 °C
?. [Preparation of reagents]
Add  MilliQ directly into the original bottle of Proteinase K, vortex to mix
25 mL
?. [Preparation of reagents]
Aliquot 600 uL to microtubes (around 45 microtubes) and keep frozen at
-20 °C
?. [Preparation of reagents]
DAPI primary stock Add  MilliQ directly into the original package and keep frozen at
2 mL
-20 °C
?. [Preliminary extraction efficiency test]
Prepare boiling bath.
?. [Preliminary extraction efficiency test]
Sonicate
?. [Preliminary extraction efficiency test]
Transfer extract to a 5 mL centrifuge tube, label the tube with number of extraction.
?. [Preliminary extraction efficiency test]
[Repeat until total extract number reaches 10.]
?. [Preliminary extraction efficiency test]
For all extract:
Centrifuge: 13300 33, 5 min
?. [Preliminary extraction efficiency test]
Load microtitre plate with  extract (triplicate).Tris buffer  is used as blank.
250 µl
?. [Preliminary extraction efficiency test]
In the dimmed room with only red light bulb on, add  DAPI to each sample in the plate.
30 µl
?. [Preliminary extraction efficiency test]
Adhere black film on the top of a microplate lid and cover the plate with this lid.
?. [Preliminary extraction efficiency test]
Read fluorescence: excitation at 410 nm and emission at 550 nm
?. [Preliminary extraction efficiency test]
Plot fluorescence intensity versus number of extraction.The number of extract (N) is the stationary point where the fluorescence of stained extract stops decreasing or the derivative of the fluorescence after that point is close to zero.
?. [Extraction of polyphosphate from samples]
Prepare boiling bath.
?. [Extraction of polyphosphate from samples]
Place vials in boiling bath
?. [Extraction of polyphosphate from samples]
Sonicate
?. [Extraction of polyphosphate from samples]
Remove extract to a centrifuge tube, and then .
[until total extract reaches N+1]
?. [Preliminary extraction efficiency test]
Keep in boiling bath.
?. [Enzyme treated extract]
Centrifuge the mixture of 1~N extract
Centrifuge: 133000 33, 5 min
?. [Enzyme treated extract]
Transfer  supernatant to a 5 mL centrifuge tube, add  RNase and  DNase
4 mL
40 µl
40 µl
?. [Enzyme treated extract]
Incubate at , shake continuously
37 °C
?. [Enzyme treated extract]
Add  Proteinase
80 µl
?. [Enzyme treated extract]
Incubate at , shake continuously.
37 °C
?. [Enzyme treated standard amended extract]
Transfer  of enzyme treated extract into a 5 mL centrifuge tube
1680 µl
?. [Enzyme treated N+1 extract]
Centrifuge extract "N+1"
Centrifuge: 13300 33, 5 min
?. [Enzyme treated N+1 extract]
Transfer   supernatant into a 2 mL tube, add  RNase and  DNase
1 mL
10 µl
10 µl
?. [Enzyme treated N+1 extract]
Incubate at , shake continuously
37 °C
?. [Enzyme treated N+1 extract]
Add  Proteinase
20 µl
?. [Enzyme treated N+1 extract]
Incubate at , shake continuously
37 °C
?. [Load microtiter plate]
Load  samples to the microplate. Organize samples as shown in the following scheme:
250 mL
?. [Load microtiter plate]
Adhere black film on the top of a microplate lid and cover the plate with this lid.
?. [Load microtiter plate]
Read fluorescence: excitation at 410 nm and emission at 550 nm
?. [Calculation]
Definition of symbol
?. [Calculation]
?. [Calculation]
?. [Preparation of reagents]
Transfer the pellet into a 100 mL graduated cylinder.
?. [Preparation of reagents]
PolyP secondary standard stockIf the pellet is far more than 10 mg, dilute primary to secondary to bring down the concentration before preparing working standard
?. [Preliminary extraction efficiency test]
Shake at room temperature
?. [Preliminary extraction efficiency test]
Add  or  Tris buffer  , sonicate.
2 mL
4 mL
?. [Preliminary extraction efficiency test]
If the sample has less than 3 ug total particulate phosphate, use  Tris Buffer  for each extraction.Otherwise, use  Tris Buffer   for each extraction.
2 mL
4 mL
?. [Extraction of polyphosphate from samples]
Add same amount of Tris buffer  as preliminary test and then sonicate
?. [Extraction of polyphosphate from samples]
Combine extract 1~N into the same tube, transfer extract N+1 into another tube.
?. [Preliminary extraction efficiency test]
Transfer sample into glass vial
?. [Extraction of polyphosphate from samples]
Transfer samples into glass vials
?. [Enzyme treated standard amended extract]
Prepare PolyP working standard Based on the actual concentration of PolyP (45) primary or secondary standard stock, dilute a certain volume of stock with  Tris buffer
1 mL
?. [Enzyme treated standard amended extract]
Add   polyP working standard to  of enzyme treated extract, vortex.
320 µl
1680 µl
?. [Load microtiter plate]
Prepare DAPI working solution Dilute  of  DAPI stock with  MilliQ in a foil wrapped 600 uL microtube and vortex.
2.1 µl
300 µl
?. [Load microtiter plate]
In a dimmed room with only red bulb on, add  DAPI working solution  to each sample in the microplate except for those labelled with UN.
30 µl
?. [Load microtiter plate]
Shake at room temperature.
?. [Preliminary extraction efficiency test]
Prepare DAPI working solution Dilute  of  DAPI stock with  MilliQ in a foil wrapped 600 uL microtube and vortex.
2.1 µl
300 µl