TITLE: Basic pipet technique training
AUTHORS: Ying-Yu Hu

[DESCRIPTION]
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. 


 





[STEPS]
SECTION: Preparing standard working solutions
1. Date:

Trainee:


SECTION: Prepare working reagents
6.  2.5 % ammonium molybdate reagent

  

SECTION: Prepare working reagents
10. Use graduated cylinder, measure and transfer 6 mL  2.5 % ammonium molybdate reagent into the sulfuric acid tube.
SECTION: Colorimetric measurement
13. Preheat incubator/shaker to 37 °C 
 

SECTION: Colorimetric measurement
14. Add  500 µL reagent to each standard working solution.
SECTION: Colorimetric measurement
15. Vortex each tube.
SECTION: Colorimetric measurement
16. Incubate at 37 °C  for 60 min  while shaking at 200 rpm







SECTION: Colorimetric measurement
17. Load microplate with 250 uL reactant from each tube, duplicate. 








SECTION: Colorimetric measurement
20.   Setup layout and method
 

SECTION: Calculation
24. 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.  
SECTION: Prepare working reagents
8.  Sulfuric acid reagent:

SECTION: Prepare working reagents
5. Need the following materials:
   SupplyCheck1Microtube rack217 X 2 mL microtubes3MilliQ water container410 uL pipet5100 uL pipet61000 uL pipet710 uL pipet tip8100 uL pipet tip91000 uL pipet tip10Vortex mixer11Sharpies12Primary stock solution of KH2PO413MilliQ


SECTION: Preparing standard working solutions
4. Standard working solutions 
 

 

SECTION: Preparing standard working solutions
3. 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. 

 

 

SECTION: Prepare working reagents
11. Use graduated cylinder, measure and transfer 6 mL  10 %  ascorbic acid reagent into the sulfuric acid tube.
SECTION: Prepare working reagents
12. Vortex the reagent.
SECTION: Colorimetric measurement
19. Turn on microplate reader
 

SECTION: Colorimetric measurement
18. Layout in the microplate:
 

SECTION: Colorimetric measurement
21. Place microplate onto the plate holder
SECTION: Colorimetric measurement
22. Read the plate
SECTION: Colorimetric measurement
23. Export data to Excel sheet
SECTION: Calculation
26. 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
 

SECTION: Review
27. What information does LOD provide?
SECTION: Prepare working reagents
6.1. In an anti-static weight plate, weigh  ~0.25 g ammonium molybdate, transfer into the labelled falcon tube.
 

SECTION: Prepare working reagents
6.2. Measure 10 mL MilliQ (use transfer pipet for the final topping)
SECTION: Prepare working reagents
6.3. Rinse the residue on the weighing plate into the falcon tube with the MilliQ.
SECTION: Prepare working reagents
6.4. Transfer all remaining MilliQ into the falcon tube
SECTION: Prepare working reagents
7. 10 % ascorbic acid reagent (avoid light exposure)

  

SECTION: Prepare working reagents
7.1. In an anti-static weight plate, weigh  ~1 g ascorbic acid, transfer into the labelled falcon tube.
 

SECTION: Prepare working reagents
7.2. Measure 9 mL MilliQ (use transfer pipet for the final topping)
SECTION: Prepare working reagents
7.3. Rinse the residue on the weighing plate into the falcon tube with the MilliQ.
SECTION: Prepare working reagents
7.4. Transfer all remaining MilliQ into the falcon tube
SECTION: Prepare working reagents
9. Vortex ammonium molybdate and ascorbic acid solutions.
SECTION: Calculation
25. 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
SECTION: Preparing standard working solutions
2. Need the following materials:
 

SECTION: Prepare working reagents
8.1. Measure 17 mL MilliQ (use transfer pipet for the final topping) and transfer into a Falcon tube
SECTION: Prepare working reagents
8.2. Carefully add 1 mL   18 M concentrated sulfuric acid into the water
SECTION: Clean-up
29. Neutralize acidic chemicals with baking soda in a plastic beaker
SECTION: Review
28. Is it necessary to be aware of the order when mixing water with concentrated sulfuric acid?
SECTION: Clean-up
30. Rinse microtubes and microplate with tap water
SECTION: Clean-up
31. Dispose microtubes and microplate into recyclable garbage bin
SECTION: Clean-up
32. Dispose pipet tips into sharp garbage container
SECTION: Clean-up
33. Rinse graduated cylinder, Falcon tubes and MilliQ container with DI water, place them into a basket to air-dry