Mar 27, 2024

Public workspaceBasic pipet technique training

DOI
dx.doi.org/10.17504/protocols.io.36wgqjo4yvk5/v1
Basic pipet technique training
  • 1Dalhousie University
Ying-Yu Hu
Open access
DOI: dx.doi.org/10.17504/protocols.io.36wgqjo4yvk5/v1
Protocol Citation: Ying-Yu Hu 2024. Basic pipet technique training. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqjo4yvk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 08, 2023
Last Modified: March 27, 2024
Protocol Integer ID: 74966
Keywords: orthophosphate
Funders Acknowledgement:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 723789
Abstract
Here we adopt the molybdate colorimetric reaction from our Total particulate phosphorus (TPP) measurement as a training material for our new lab members to learn basic pipetting technique.


CITATION
YY Hu, AJ Irwin, ZV Finkel (2022). Improving quantification of particulate phosphorus. Limnology and Oceanography: Methods.
LINK
10.1002/lom3.10517




Protocol materials
ReagentAmmonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G
Step 6
ReagentAscorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G
Step 7
Safety warnings
Attention
Acidic reagents need to be neutralized before dumped into the sink.
Preparing standard working solutions
Preparing standard working solutions
2h
Date:

Trainee:


Need the following materials:
SupplyCheck
Microtube rack
17 X 2 mL microtubes
MilliQ water container
10 uL pipet
100 uL pipet
1000 uL pipet
10 uL pipet tip
100 uL pipet tip
1000 uL pipet tip
Vortex mixer
Sharpies
Primary stock solution of KH2PO4
MilliQ

How to aspire and dispense with pipet

(a) When aspiring solution, ensure the pipette to be held vertically.
(b) When dispensing, ensure you hold the pipette at an angle (10-45°). Working to these angles ensures the desired liquid amount is drawn into the tip properly and that all of the liquid is fully dispensed without leaving any residue in the tip.

https://www.americanlaboratory.com/914-Application-Notes/240482-Ten-Tips-for-Proper-Pipetting

Note
Always pay attention to the pipet tip, check if there is air bubble in the tip or slightly leaking on the tip.

Standard working solutions
Concentration of KH2PO4 primary stockUnit

KH2PO4Primary (uL)CheckMilliQ (uL)Check
S10500
S22498
S35495
S410490
S525475
S650450
S775425
S8100400

Prepare working reagents
Prepare working reagents
2h
Need the following materials:
SupplyCheck
50 mL Falcon tube (3)
Transfer pipet (1)
Scoopula (1)
Anti-static weighting plate (2)
MilliQ
Kimwipe
Chamber balance
Graduated cylinder (1X25 mL)
Goggles
Concentration2.5 % ammonium molybdate reagent

ReagentAmmonium molybdateSigma AldrichCatalog #09878-100G

In an anti-static weight plate, weigh ~0.25 g ammonium molybdate, transfer into the labelled falcon tube.
Actual weight (g)

Measure 10 mL MilliQ (use transfer pipet for the final topping)
Rinse the residue on the weighing plate into the falcon tube with the MilliQ.
Transfer all remaining MilliQ into the falcon tube
Concentration10 % ascorbic acid reagent (avoid light exposure)

ReagentAscorbic acidSigma AldrichCatalog #A5960-100G

In an anti-static weight plate, weigh ~1 g ascorbic acid, transfer into the labelled falcon tube.
Actual weight (g)

Measure 9 mL MilliQ (use transfer pipet for the final topping)
Rinse the residue on the weighing plate into the falcon tube with the MilliQ.
Transfer all remaining MilliQ into the falcon tube
Sulfuric acid reagent:

Measure 17 mL MilliQ (use transfer pipet for the final topping) and transfer into a Falcon tube
Carefully add Amount1 mL Concentration18 Mass Percent concentrated sulfuric acid into the water
Vortex ammonium molybdate and ascorbic acid solutions.
Use graduated cylinder, measure and transfer 6 mL Concentration2.5 % ammonium molybdate reagent into the sulfuric acid tube.
Use graduated cylinder, measure and transfer 6 mL Concentration10 % ascorbic acid reagent into the sulfuric acid tube.
Vortex the reagent.
Colorimetric measurement
Colorimetric measurement
2h
Preheat incubator/shaker to Temperature37 °C
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU

Add Amount500 µL reagent to each standard working solution.
Vortex each tube.
Incubate at Temperature37 °C for Duration01:00:00 while shaking at 200 rpm







1h
Load microplate with 250 uL reactant from each tube, duplicate.








Layout in the microplate:
1234
AS1S1S1S1
BS2S2S2S2
CS3S3S3S3
DS4S4S4S4
ES5S5S5S5
FS6S6S6S6
GS7S7S7S7
HS8S8S8S8

Turn on microplate reader
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU

Setup layout and method
AB
Shake duration00:00:05
Shaking typeContinuous
Shaking forceHigh
Shaking speed [rpm]600
Wavelength [nm]820
Measurement Time [ms]100

Place microplate onto the plate holder
Read the plate
Export data to Excel sheet
Calculation
Calculation
In the Excel sheet, subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other standard working solutions.

This gives the corrected absorbance from each well.
Calculate the concentration of each standard working solution
Unit for each content:
Primary concentration g/L
Primary volume uL
MilliQ volume uL
Standard working solution g/L
Phosphate in standard working solution uM
Y-axis: Corrected absorbance
X-axis: Phosphate (uM)
Calculate slope (S), intercept (b), coefficient of determination (R2), standard deviation (Sy) of the absorbance from the four replicates of the blank, and LOD = 3.3 X Sy/S
ParameterValue
S
b
R2
Sy
LOD

Review
Review
What information does LOD provide?
Is it necessary to be aware of the order when mixing water with concentrated sulfuric acid?
Clean-up
Clean-up
Neutralize acidic chemicals with baking soda in a plastic beaker
Rinse microtubes and microplate with tap water
Dispose microtubes and microplate into recyclable garbage bin
Dispose pipet tips into sharp garbage container
Rinse graduated cylinder, Falcon tubes and MilliQ container with DI water, place them into a basket to air-dry
Citations
YY Hu, AJ Irwin, ZV Finkel. Improving quantification of particulate phosphorus
10.1002/lom3.10517