Basic pipet technique training

Ying-Yu Hu

Mar 27, 2024

Version 1

Basic pipet technique training V.1

DOI

https://dx.doi.org/10.17504/protocols.io.36wgqjo4yvk5/v1

Ying-Yu Hu¹

¹Dalhousie University

Marine Microbial Macroecology Lab
Tech. support email: [email protected]

Ying-Yu Hu

Dalhousie University

DOI: https://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, total particulate phosphorus, basic pipet technique training, basic pipetting technique, molybdate colorimetric reaction, tpp

Funders Acknowledgements:

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.10.1002/lom3.10517
LINK

Protocol materials

ReagentAmmonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G 
ReagentAscorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G 

Safety warnings

Acidic reagents need to be neutralized before dumped into the sink.

Preparing standard working solutions

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

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 molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #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 acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #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

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

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

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

What information does LOD provide?

Is it necessary to be aware of the order when mixing water with concentrated sulfuric acid?

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

	Supply	Check
	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

KH2PO4	Primary (uL)	Check	MilliQ (uL)	Check
S1	0		500
S2	2		498
S3	5		495
S4	10		490
S5	25		475
S6	50		450
S7	75		425
S8	100		400

	Supply	Check
	50 mL Falcon tube (3)
	Transfer pipet (1)
	Scoopula (1)
	Anti-static weighting plate (2)
	MilliQ
	Kimwipe
	Chamber balance
	Graduated cylinder (1X25 mL)
	Goggles

	1	2	3	4
A	S1	S1	S1	S1
B	S2	S2	S2	S2
C	S3	S3	S3	S3
D	S4	S4	S4	S4
E	S5	S5	S5	S5
F	S6	S6	S6	S6
G	S7	S7	S7	S7
H	S8	S8	S8	S8

	A	B
	Shake duration	00:00:05
	Shaking type	Continuous
	Shaking force	High
	Shaking speed [rpm]	600
	Wavelength [nm]	820
	Measurement Time [ms]	100

	Parameter	Value
	S
	b
	R2
	Sy
	LOD

	1	2	3	4
A	S1	S1	S1	S1
B	S2	S2	S2	S2
C	S3	S3	S3	S3
D	S4	S4	S4	S4
E	S5	S5	S5	S5
F	S6	S6	S6	S6
G	S7	S7	S7	S7
H	S8	S8	S8	S8

Public workspaceBasic pipet technique training V.1

Basic pipet technique training V.1

	1	2	3	4
A	S1	S1	S1	S1
B	S2	S2	S2	S2
C	S3	S3	S3	S3
D	S4	S4	S4	S4
E	S5	S5	S5	S5
F	S6	S6	S6	S6
G	S7	S7	S7	S7
H	S8	S8	S8	S8