Technote For Volumetric Studies

     


  Detailed Assay Method To Determine Volumetric Liquid Transfer With Pin Tools
  Using Fitc In 100% DMSO

 
 


I.  OVERVIEW:

FITC Transfer Summary: 

  Dilutions of FITC in 100% DMSO are aliquoted to a polypropylene Source Plate.
  200 ul 1 M Tris-HCl, pH 8.0 is added to each well of a solid black Assay Plate.
  Pin tool is dipped into FITC Source Plate, and then transferred to Assay Plate.
  Pin tool is moved up and down 3X to mix FITC with Tris.
  Cover and store at room temperature in the dark until ready to read plates.
  Mix the well contents on a vibrator or let sit over night to allow for complete mixing.
  Read at 520 nm using Tris-HCL as blank.

 

CONTROLS 

A standard curve is generated with two-fold serial dilutions of FITC in DMSO using a multi-channel pipettor.  In this case, 10 ul of the appropriate serial dilutions of FITC are added to 190 ul Tris in a separate Assay Plate with a 12-channel pipettor.  The slope and y-intercept values from the linear range of the standard curve are used to determine the mean volume of FITC transferred by each pin tool.

 

DETERMINATION OF STANDARD CURVE DILUTION RANGE 

To determine the best dilutions of FITC to use for the control standard curve, an initial test should be run with two-fold serial dilutions of FITC (we used 24 two-fold serial dilutions initially).  Transfer 10 ul of each FITC dilution to 190 ul of Tris with a 12-channel pipettor.  This will determine the best range, over 12 dilutions, to use as the control standard curve.  In addition, the appropriate dilution range for each pin tool can be extrapolated from this curve. 

 

STOCK CONCENTRATION OF FITC IN SOURCE PLATES

 

CONTROL SOURCE PLATE: 

For the control standard curve, twelve two-fold serial dilutions of FITC in 100% DMSO are prepared and added to a row of the Control Source Plate (See Section IV).  The stock concentrations range from 0.156 to 0.000076 mg/ml in this example.  Using a 12-channel pipettor, 10 ul of each FITC dilution is added to 190 ul Tris in seven rows of the Control Assay Plate.  The last row contains 200 ul of Tris as a blank control.

 

 

PIN TOOL SOURCE PLATE: 

For each pin tool, two different eight-fold dilutions should be tested.  Approximately 20 mls each of the two dilutions is needed to fill each Pin Tool Source Plate (See Section IV).  The final volume in each well should be 200 ul.  One Pin Tool Source Plate can be used for multiple pin tools.  The table below lists the average FITC concentration used for testing the following pin tools:  

 

 

 

Pin Tool

FITC Concentration in

Pin Tool Source Plates (mg/ml)

Dilution 1

Dilution 2

FP9

10

1.25

FP1

1.25

0.1563

FP1S6

1.25

0.1563

FP1S10

1.25

0.1563

FP1S50

0.1563

0.01953

FP3

0.1563

0.01953

FP3S100

0.1563

0.01953

FP3S200

0.1563

0.01953

FP3S500

0.1563

0.01953

 

FINAL CONCENTRATION OF FITC IN ASSAY PLATES 

In our initial testing, we found that a good, overall final concentration for FITC in the standard curve assay plate ranged from 0.0078 to 0.0000038 mg/ml in two-fold serial dilutions across the 12 wells of the plate.  For testing the pin tools with an entire plate of a single dilution, FITC concentrations were selected such that the final concentration of FITC in the Pin Tool Assay Plate was between 0.000244 and 0.000061 mg/ml. 

 

CALCULATION OF VOLUME TRANSFERRED BY PIN TOOL 

To calculate the volume of FITC transferred in each assay:

  1. Read each assay plate at A520 (We read at A535, because that is what the instrument that is available to us is set at.  A535 is within the emission peak of FITC).
     

  2. Calculate, and then plot, the mean values from all of the control standard curves.
     

  3. Calculate the slope, y-intercept and R2-Value from the mean values in the linear range of the control standard curve.
     

  4. For each assay plate, calculate the CV% and mean A520 of each dilution from the pin tool.
     

  5. Identify the A520 values from the pin tool that fall within the linear range of the control standard curve (y).
     

  6. Using the slope and y-intercept (m and b) from the control standard curve mean values and the A520 value that fall within the linear range from the pin tool transfer (y), the number of mg of FITC (x) in that pin tool dilution are calculated.
     

  7. Based on the starting concentration of FITC in that dilution, the number of hl transferred by the pin tool are calculated.

 

II.  REAGENTS: 

ORDERING AND STORAGE INFORMATION: 

 

Name

Description

Sigma    Part No.

Quantity

Storage

FITC

Fluorescein 5-isothiocyanate, Isomer I

F7250

50-500 mg

2-8oC

DMSO

Dimethyl Sulfoxide

 

D2650

100 ml

RT

Tris

Tris-HCL, 1 M Stock Solution, pH 8.0

T3038

1 liter

RT

 

  

III.  PREPARATION OF STOCK SOLUTIONS:

10 mg/ml FITC in 100% DMSO:
   
Add 50 ml fresh 100% DMSO to 500 mg FITC
    Mix well
    Store @ RT, covered with foil

 

 

IV.  PREPARATION OF FITC SERIAL DILUTIONS:

A total of 500 ul of each two-fold dilution is prepared for the Control Standard Curve Source Plate.  The table below is a guideline for preparing the Control Standard Curve dilutions. 

1:2 Serial Dilutions for Control Standard Curve

~500 ul Final Volume Each

   
Diln #

   
Initial Vol (ul)


Initial Conc (mg/ml)

Diluent DMSO Vol (ul)

 
Final Vol (ul)


Final Conc (mg/ml)

1

125

1.25

875

1000

0.156250

2

500

0.156250

500

1000

0.078125

3

500

0.078125

500

1000

0.039063

4

500

0.039063

500

1000

0.019531

5

500

0.019531

500

1000

0.009766

6

500

0.009766

500

1000

0.004883

7

500

0.004883

500

1000

0.002441

8

500

0.002441

500

1000

0.001221

9

500

0.001221

500

1000

0.000610

10

500

0.000610

500

1000

0.000305

11

500

0.000305

500

1000

0.000153

12

500

0.000153

500

1000

0.000076

A total of 19.2 mls each of the two dilutions is needed to fill each Pin Tool Source Plate.  The table below can be used as a guideline for preparing the dilutions, including extra for the Control Standard Curve and pipetting variables. 

1:8 Serial Dilutions for Pin Tool Source Plates

~21 ml Final Volume Each

   
Diln #

   
Initial Vol (ml)


Initial Conc (mg/ml)

Diluent DMSO Vol(ml)


Final Vol (ml)


Final Conc (mg/ml)

1

 

 

 

24

10

2

3

10

21

24

1.25

3

3

1.25

21

24

0.1563

4

3

0.1563

21

24

0.0195

 


V.  ASSAY PREPARATION:

  1. Calculate volume of reagents needed:
    1. FITC Stock Solution (10 mg/ml)
    2. DMSO
    3. 1 M Tris-HCl, pH 8.0
  2. Check reagents.
  3. Prepare labels for plates.
            e.g., 010508-1 FP9N-L2L
            = yr/mo/day-plate #  pin tool/coating (hydrophobic or not)-type of transfer
            Sample plate labels for one assay:
                    010508-1 FP9N-L2L
                    010508-2 FP9N-M2L
                    010508-3 FP9N-L2D
                    010508-4 FP9N-D2L
                    010508-5 FP9H-L2L
                    010508-6 FP9H-M2L
                    010508-7 FP9H-L2D
                    010508-8 FP9H-D2L
  1. Label plates.
  2. Label tubes for FITC serial dilutions.
  3. Aliquot appropriate volume of DMSO to dilution tubes.
  4. Prepare eight-fold Pin Tool Source Plate dilutions in 50 ml polypropylene tubes
  5. Prepare two-fold Standard Curve Source Plate dilutions in microfuge tubes.
  6. Aliquot appropriate dilutions to Control and Pin Tool Source Plates.
    1. Aliquot 200 ul of the appropriate dilution to each well of the Pin Tool Source Plate (two dilution plates per pin tool).
    2. Aliquot 200 ul of each two-fold Standard Curve dilutions to one row of the Control Source Plate
  7. Aliquot appropriate volumes of 1 M Tris-HCl, pH 8.0 to Assay Plates.
    1. 200 ul to each well of every Pin Tool Assay Plate, except Dry Plate touch-off Assay Plate (L2D).
    2. 190 ul to each well in rows A-G of the Control Assay Plate.
    3. 200 ul to each well in row H of the Control Assay Plate.
  8. Set up wash stations.
  9. Set pipettors at proper settings.
  10. Set up membrane blotting stations.
  11. START ASSAY

 

  VI.  FITC PIN TOOL ASSAY:

Conditions to Test:
   
    
Liquid-to-Liquid
          Liquid-to-Membrane
          Liquid-to-Dry-Plate

Liquid-to-Liquid (L2L)
Will measure total amount of FITC transferred by pins, including sides and hanging drop or slot.

  1. Prepare two L2L Assay Plates.
        a.    Aliquot 200 ul Tris/well to each well of Pin Tool L2L Assay Plates.

  2. Dip replicator into Pin Tool Source Plate - Dilution 1.

  3. Transfer replicator to L2L Assay Plate.

  4. Mix up and down 3X.

  5. Rinse replicator:  2X H2O, 1X Isopropanol, dry.

  6. Dip replicator into Pin Tool Source Plate - Dilution 2.

  7. Transfer replicator to second L2L Assay Plate.

  8. Mix up and down 3X.

  9. Rinse replicator:  2X H2O, 1X Isopropanol.

  10. Cover plates (with adhesive plate sealers) and store at room temperature in the dark.

  11. Mix the well contents on a vibrator or let sit over night to allow for complete mixing.

Liquid-to-Membrane/Membrane to Liquid (M2L)
Will measure total liquid transferred by sides only:
        Liquid in hanging drop or slot will be transferred to nylon membrane (not directly measured).
        Volume carried by sides of pin (M2L) is measured.

  1. Prepare Assay Plates:
        a.    Aliquot 200 ul Tris/well to each well of Pin Tool M2L Assay Plates.

  2. Dip replicator into Pin Tool Source Plate - Dilution 1.

  3. Touch off replicator pins on membrane.

  4. Transfer replicator to M2L Assay Plate.

  5. Mix up and down 3X.

  6. Rinse replicator:  2X H2O, 1X Isopropanol, dry.

  7. Dip replicator into Pin Tool Source Plate - Dilution 2.

  8. Touch off replicator pins on membrane.

  9. Transfer replicator to second M2L Assay Plate.

  10. Mix up and down 3X.

  11. Rinse replicator:  2X H2O, 1X Isopropanol.

  12. Cover plates (with adhesive plate sealers) and store at room temperature in the dark.

  13. Mix the well contents on a vibrator or let sit over night to allow for complete mixing.

Liquid-to-Dry Plate and Dry Plate-to-Liquid  (L2D & D2L)
Will measure liquid transferred from hanging drop or slot to dry plate and liquid transferred by sides and residual hanging drop:
    Volume left behind on dry plate-touch off (L2D)
    Volume remaining on pin tip and carried by sides of pin (D2L)

  1. Prepare two Assay Plates
        a.    Have Dry Assay Plate ready.
        b.    Aliquot 200 ul Tris/well to each well of Pin Tool D2L Assay Plates.

  2. Dip replicator into Pin Tool Source Plate – Dilution 1.

  3. Touch off replicator pins on Dry Assay Plate (L2D).

  4. Immediately transfer replicator to D2L Assay Plate.

  5. Mix up and down 3X.

  6. Rinse replicator:  2X H2O, 1X Isopropanol, dry.

  7. Dip replicator into Pin Tool Source Plate – Dilution 2.

  8. Touch off replicator pins on second Dry Assay Plate (L2D).

  9. Immediately transfer to second D2L Assay Plate.

  10. Mix up and down 3X.

  11. Rinse replicator:  2X H2O, 1X Isopropanol.

  12. Aliquot 200 ul Tris to each well of the two Dry Assay Plates (L2D).

  13. Cover plates (with adhesive plate sealers) and store at room temperature in the dark.

Control Standard Curve Plate
Will be used to generate mean standard curve values.  The slope and y-intercept from these values will be used to determine the mean volume of FITC transferred by each pin tool.

  1. Prepare Assay Plate:
        a.    Aliquot 190 ul Tris/well to each well in rows A-G of Control Assay Plate.
        b.    Aliquot 200 ul Tris/well to each well in row H of Control Assay Plate.

  2. Using a 12-channel pipettor, transfer 10 ml of each two-fold dilution from the Control Source Plate to the rows A-G of the Control Assay Plate.

  3. Pipet each up and down 2-3 times to mix after addition.

  4. Cover plate (with adhesive plate sealer) and store at room temperature in the dark.

  More Pin Tool Data And Charts


  • DETAILED ASSAY METHOD TO DETERMINE VOLUMETRIC LIQUID TRANSFER WITH PIN TOOLS USING HRP IN TBS/TWEEN 20
    TECHNICAL NOTE 66

I.  OVERVIEW:

HRP Reaction Summary: 

  Serial Dilutions of HRP in TBS/Tween 20 are aliquoted to a Source Plate.
  200 ul Activated OPD/Citrate Substrate Solution is added to each well of Assay Plate.
  Pin tool is dipped into HRP Source Plate, then transferred to an Assay Plate.
  Pin tool is moved up and down 3X to mix HRP with Substrate.
  Incubate 30 min at room temperature, in the dark.
  Stop reaction by addition of 100 ul of 2 M H2SO4.
  Final volume:  300 ul.
  Read at 490 nm using TBS/Tween 20 as blank.

 

CONTROLS

A standard curve is generated with two-fold serial dilutions of HRP in TBS/Tween 20 using a multi-channel pipettor.  In this case, 10 ul of the appropriate serial dilutions of HRP are added to 190 ul Substrate in a separate Assay Plate with a 12-channel pipettor.  The slope and y-intercept values from the linear range of the standard curve are used to determine the mean volume of HRP transferred by each pin tool. 

 

DETERMINATION OF STANDARD CURVE DILUTION RANGE

To determine the best dilutions of HRP to use for the control standard curve, an initial test should be run with two-fold serial dilutions of HRP (we used 24 two-fold serial dilutions initially).  Transfer 10ull of each HRP dilution to 190 ul of Substrate with a 12-channel pipettor.  Incubate the reactions for 30 minutes in the dark.  Stop the reactions with the addition of 100 ul of H2SO4. This will determine the best range, over 12 dilutions, to use as the Control Standard Curve.  In addition, the appropriate dilution range for each pin tool can be extrapolated from this curve. 

 

STOCK CONCENTRATION OF HRP IN SOURCE PLATES

CONTROL SOURCE PLATE:  Once the appropriate two-fold serial dilutions have been prepared, 200 ul of each dilution is added to Row A of the Control Source Plate.  In this example, the stock concentrations range from 2.54 to 0.00125 U/ml in two-fold serial dilutions across the Control Source Plate.  

PIN TOOL SOURCE PLATE:  For each pin tool, the appropriate range of dilutions needs to be calculated.  Once the appropriate two-fold serial dilutions have been prepared, 200 ul of each dilution is added to Columns 1-12 of the Pin Tool Source Plate.  As an example, the FP1 pin tools will transfer approximately 20 nl.  Therefore, the stock concentrations of HRP range from 1,310 to 0.64 U/ml in two-fold serial dilutions across the Pin Tool Source Plate.  Several pin tools may use the same range of dilutions.   

With this format, usually two dilutions on every pin tool transfer will fall within the linear range of the control standard curve.

  

FINAL CONCENTRATION OF HRP IN ASSAY PLATES

In our initial testing, we found that a good, overall final concentration for HRP in the assay plates ranged from 0.127 to 0.0000625 U/ml in two-fold serial dilutions across the 12 wells of the Pin Tool Assay Plate, regardless of the transfer tool. 

 

CALCULATION OF VOLUME TRANSFERRED BY PIN TOOL

 To calculate the volume of HRP transferred in each assay:

  1. Read each assay plate at A490.
  2. Calculate, and then plot, the mean values from all of the control standard curves.
  3. Calculate the slope, y-intercept and R2-Value from the mean values in the linear range of the control standard curve.
  4. For each assay plate, calculate the CV% and mean A490 of each dilution from the pin tool.
  5. Identify the A490 values from the pin tool that fall within the linear range of the control standard curve (y).
  6. Using the slope and y-intercept (m and b) from the control standard curve mean values and the A490 value that fall within the linear range from the pin tool transfer (y), the number of units of HRP (x) in that pin tool dilution are calculated.
  7. Based on the starting concentration of HRP in that dilution, the number of hl transferred by the pin tool are calculated.

 

II.  REAGENTS:

 ORDERING AND STORAGE INFORMATION: 

Name

Description

Sigma    Part No.

Quantity

Storage

OPD

o-Phenylenediamine Tablets, Dihydrochloride

20 mg substrate per tablet

P7288

50 Tablets

2-8oC

HRP

Horseradish Peroxidase

~1000 U/mg

 

P6782

10 mg

<0oC

Citric Acid

Free Acid, Anhydrous

 

 

C0759

100 g

RT

Sodium Phosphate

Heptahydrate

Na2HPO4.7H2O

 

S9390

100 g

RT

Hydrogen Peroxide

30% H2O2

 

 

H0904

100 ml

2-8oC

Hydrochloric Acid

HCL

1.0 N

 

35328

1 L

RT

Sulfuric Acid

H2SO4

For prep of 1L 0.5M

 

38294

0.5 M

RT

 

 

III.  PREPARATION OF STOCK SOLUTIONS:

Citrate Buffer:

0.1 M Citric Acid Solution
9.6 g Citric Acid (anhydrous)
to 500 ml w/ distilled H2O
Store @ RT

0.2 M Na2HPO4 Solution
26.87 g Na2HPO4.7H2O
to 500 ml w/ distilled H2O
Store @ RT

Citrate Buffer:
486 ml 0.1 M Citric Acid 
500 ml 0.2 M Na2HPO4
        Mix
        pH to 5.0 w/ 1.0 N HCl
        Filter sterilize
        Store @ RT

10X o-Phenylenediamine (OPD) Solution
USE CAUTION – POSSIBLE CARCINOGEN

Add     400 mg of o-phenylenediamine (OPD) (20 x 20 mg OPD Tablets)
            100 ml citrate buffer 
Mix
Store in 2 ml aliquots @ -20oC
            Add 2 ml to 1-dram vials
            Store @ -20oC in the dark.
            This will make ~50 1 dram vials.

Activated OPD/Citrate Substrate Solution
Just before use, add 2 ml of 10X OPD to 18 ml citrate buffer.
Then activate by adding 4 ml of 30% H2O2.
Final Concentration of Substrate Solution:
            0.05 M citrate
            0.1 M phosphate buffer, pH 5
            0.04% o-phenylenediamine
            0.006% H2O2

IV.  PREPARATION OF HRP SERIAL DILUTIONS:

A total of 1800 ul of each dilution in the standard curve is needed for the two Source Plates: 
1600 ul of each dilution is needed for the Pin Tool Source Plate (200 ul/well, 8 wells/dilution) and approximately 200 ul of each dilution in the Control Source Plate (1 well/dilution).  Prepare 2.0 ml (N+1) of each dilution. 

AQUEOUS STANDARD CURVE DILUTIONS IN FALCON TUBES

Dilution 
Number                           Dilution                                               Final Volume/Concentration

  1.        200 ul 26.2 U/ul HRP + 3800 ul TBS/Tween20          = 4.0 ml 1.31 U/ul HRP
  2.        2.0 ml 1.31 U/ul HRP + 2.0 ml TBS/Tween20               = 4.0 ml 0.655 U/ul HRP
  3.        2.0 ml 0.655 U/ul HRP + 2.0 ml TBS/Tween20             = 4.0 ml 0.3275 U/ul HRP

    ..............                                                                            
    21                                                                                                     = 4.0 ml 0.00000125 U/ul HRP

V.  PREPARATION OF SUBSTRATE AND H2SO4:

         Calculate OPD/Citrate Substrate needed:
            4 plates per pin tool:
            @200 ul per well, 19.2 ml per plate or 76.8 ml per pin tool
             Prepare 80 ml Activated OPD/Citrate Substrate per pin tool.

          Calculate H2SO4 needed:
            @100 ul per well, 9.6 ml per plate or 38.4 ml per pin tool
             Prepare 40 ml 2 N H2SO4 per pin tool. 

VI.  ASSAY PREPARATION:

  1. Calculate volume of reagents needed:
    1. HRP
    2. 10X OPD
    3. Citrate Buffer
    4. Hydrogen Peroxide
    5. H2SO4
  2. Check reagents.
  3. Prepare labels for plates, e.g., 010508-1 FP9N-L2L
                = yr/mo/day-plate #  pin tool/coating (hydrophobic or not)-type of transfer
                Sample plate labels for one assay:
                        010508-1 FP9N-L2L
                        010508-2 FP9N-M2L
                        010508-3 FP9N-L2D
                        010508-4 FP9N-D2L
                        010508-5 FP9H-L2L
                        010508-6 FP9H-M2L
                        010508-7 FP9H-L2D
                        010508-8 FP9H-D2L
  1. Label plates.
  2. Label tubes for HRP serial dilutions.
  3. Aliquot appropriate volume of TBS/Tween 20 diluent to serial dilution tubes.
  4. Thaw appropriate number of 10X OPD aliquots.
    1. Per pin tool:
      i.     Thaw 4 x 2 ml aliquots of 10X OPD.
      ii.    Aliquot 72 ml Citrate buffer to flask.
  1. Prepare standard curve in Falcon Tubes.
  2. Aliquot appropriate standard curve dilutions to Pin Tool and Control Source Plates.
    1. Aliquot 200 ul Dilutions10-21 to Row A of Control Source Plate.
    2. Aliquot 200 ul Dilutions 1-12 to Rows A through H of Pin Tool Source Plate. 
    3. See Figure 1 for diagram of Source Plates.
  3. Prepare activated OPD/Citrate Substrate in flask.
    1. Per pin tool:
          8 ml 10X OPD
          72 ml Citrate Buffer
          16 ul H2O2
  1. Aliquot appropriate volumes of activated OPD/Citrate Substrate to Assay Plates.
    1. 190 ul to Row A-G of Control Source Plate.
    2. 200 ul to Row H of the Control Source Plate
    3. 200 ul to Rows A-H of the Pin Tool Source Plates.
  2. Set up wash stations.
  3. Set pipettors at proper settings.
  4. Set up membrane blotting stations.
  5. Have clock/timer nearby.
  6. Print table for logging start and stop times of each plate.
  7. START ASSAY

 

VII.  HRP PIN TOOL ASSAY:
   
    Conditions to Test:
   
            Liquid-to-Liquid
                Liquid-to-Membrane
                Liquid-to-Dry Plate

Liquid-to-Liquid (L2L)
Will measure total amount of HRP transferred by pins, including sides and hanging drop or slot (L2L).

  1. Prepare L2L Assay Plate.
        a.    Aliquot 200 ul Substrate/well to L2L Assay Plate.

  2. Dip replicator into Pin Tool Source Plate.

  3. Transfer replicator to L2L Assay Plate.

  4. Mix up and down 3X.

  5. Start incubation time.

  6. Rinse replicator:  2X H2O, 1X Isopropanol.

  7. Incubate 30 minutes, RT, in the dark.

  8. Stop reaction by addition of 100 ul H2SO4.

Liquid-to-Membrane/Membrane to Liquid (M2L)
Will measure total liquid transferred by sides only:
        Liquid in hanging drop or slot will be transferred to nylon membrane (not directly measured).
        Volume carried by sides of pin (M2L).

  1. Prepare M2L Assay Plate:
        a.       Aliquot 200 ml Substrate/well to M2L Assay Plate.

  2. Dip replicator into Pin Tool Source Plate.

  3. Touch off replicator pins on membrane.

  4. Transfer replicator to M2L Assay Plate.

  5. Mix up and down 3X.

  6. Start incubation time.

  7. Rinse replicator:  2X H2O, 1X Isopropanol.

  8. Incubate 30 minutes, RT, in the dark.

  9. Stop reaction by addition of 100 ul H2SO4.

Liquid-to-Dry Plate and Dry Plate-to-Liquid (L2D & D2L)
Will measure liquid transferred from hanging drop or slot to dry plate and liquid transferred by sides:
        Volume transferred to dry plate (L2D).
        Volume remaining on pin tip and carried by sides of pin (D2L).

  1. Prepare two Assay Plates:
        a.       Have the L2D-Dry Assay Plate ready.
        b.      Aliquot 200 ul Substrate/well to D2L Assay Plate.

  2. Dip replicator into Pin Tool Source Plate.

  3. Touch off replicator pins on Dry Assay Plate (L2D).

  4. Immediately transfer to D2L Assay Plate.

  5. Mix up and down 3X.

  6. Start incubation time.

  7. Add 200 ul Substrate to each well in Dry Assay Plate (L2D).

  8. Start incubation time for L2D Plate.

  9. Rinse replicator:  2X H2O, 1X Isopropanol.

  10. Incubate each plate 30 minutes, RT, in the dark.

  11. Stop reaction by addition of 100 ul H2SO4.

Control Standard Curve Plate

Will be used to generate mean standard curve values.  The slope and y-intercept from these values will be used to determine the mean volume of HRP transferred by each pin tool.

  1. Prepare Control Assay Plate:
        a.      Aliquot 190 ul Substrate/well to each well in rows A-G of Control Assay Plate.
        b.      Aliquot 200 ul Substrate/well to each well in row H of Control Assay Plate.

  2. Using a 12-channel pipettor, transfer 10 ml of each two-fold dilution from the Control Source Plate to the rows A-G of the Control Assay Plate.

  3. Pipet each up and down 3 times to mix after addition.

  4. Start incubation time.

  5. Last row is Substrate Only (background).

  6. Incubate 30 minutes, RT, in the dark.

  7. Stop reaction by addition of 100 ul H2SO4.

 


 

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