Basic pipet technique training
Ying-Yu Hu
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.
Before start
Steps
Preparing standard working solutions
Date:
Trainee:
Need the following materials:
| A | B |
|---|---|
| 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.
Standard working solutions
| A | B |
|---|---|
| A | B | C | D | E |
|---|---|---|---|---|
| S1 | 0 | 500 | ||
| S2 | 2 | 498 | ||
| S3 | 5 | 495 | ||
| S4 | 10 | 490 | ||
| S5 | 25 | 475 | ||
| S6 | 50 | 450 | ||
| S7 | 75 | 425 | ||
| S8 | 100 | 400 |
Prepare working reagents
Need the following materials:
| A | B |
|---|---|
| 50 mL Falcon tube (3) | |
| Transfer pipet (1) | |
| Scoopula (1) | |
| Anti-static weighting plate (2) | |
| MilliQ | |
| Kimwipe | |
| Chamber balance | |
| Graduated cylinder (1X25 mL) | |
| Goggles |
2.5% ammonium molybdate reagent
In an anti-static weight plate, weigh ~0.25 g ammonium molybdate, transfer into the labelled falcon tube.
| A |
|---|
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
10% ascorbic acid reagent (avoid light exposure)
In an anti-static weight plate, weigh ~1 g ascorbic acid, transfer into the labelled falcon tube.
| A |
|---|
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 1mL 18 concentrated sulfuric acid into the water
Vortex ammonium molybdate and ascorbic acid solutions.
Use graduated cylinder, measure and transfer 6 mL 2.5% ammonium molybdate reagent into the sulfuric acid tube.
Use graduated cylinder, measure and transfer 6 mL 10% ascorbic acid reagent into the sulfuric acid tube.
Vortex the reagent.
Colorimetric measurement
Preheat incubator/shaker to 37°C
Equipment
| Value | Label |
|---|---|
| SHAKING INCUBATOR | NAME |
| 71L | TYPE |
| Corning® LSE™ | BRAND |
| 6753 | SKU |
Add 500µL reagent to each standard working solution.
Vortex each tube.
Incubate at 37°C for 1h 0m 0s while shaking at 200 rpm
Load microplate with 250 uL reactant from each tube, duplicate.
Layout in the microplate:
| A | B | C | D | E |
|---|---|---|---|---|
| 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 |
Turn on microplate reader
Equipment
| Value | Label |
|---|---|
| Varioskan LUX Multimode Microplate Reader | NAME |
| Thermo Fisher | BRAND |
| VL0L00D0 | SKU |
Setup layout and method
| A | B |
|---|---|
| Shake duration | 00:00:05 |
| Shaking type | Continuous |
| Shaking force | High |
| 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 S y/ S
| A | B |
|---|---|
| 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
