Jan 08, 2023

Public workspacePipetting V.1

This protocol is a draft, published without a DOI.
Pipetting
  • Carlos Goller1,
  • Carly Sjogren1
  • 1North Carolina State University
Nidhi Grover
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Protocol Citation: Carlos Goller, Carly Sjogren 2023. Pipetting . protocols.io https://protocols.io/view/pipetting-chkft4tn
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: In development
We are still developing and optimizing this protocol
Created: October 08, 2022
Last Modified: February 29, 2024
Protocol Integer ID: 71015
Keywords: Pipetting, using lab micropipette, goals lab micropipette, lab micropipette, small volumes with micropipette, micropipette, pipetting, microliter, lab skill, milliliter, small volumes of liquid, following lab skill, small volume, lab safety, ml, lab, liquid
Abstract
Overview and Goals
Lab micropipettes allow us to accurately transfer small volumes of liquids. Units to measure small volumes with micropipettes are: microliter (uL) and milliliter (mL). There are 1000 uL in 1 mL). Effectively using lab micropipettes is a valuable skill that takes practice! We will work together to learn and improve our pipetting to improve accuracy.

After completing this lab you will gain the following lab skills:

  • Lab safety and proper personal protective equipment (PPE)
  • Pipetting with micropipettes

Guidelines

Reducing waste - micropipette tips - Pipetting best practices dictate that a new micropipette tip should be used every time you pipette a new volume of liquid. This can be very important in avoiding contamination in molecular biology. Unfortunately, this also creates a considerable amount of plastic waste. For this activity, to reduce unnecessary waste, we will reuse tips when only pipetting water.
Materials
  • One 2-20 μl micropipette (p20)
  • Micropipette tips for p20
  • One Pipette Practice Card from miniPCR bio
  • Chem wipes
  • 200 μl water (tap water is fine)
  • 80 μl blue micropipetting practice fluid
  • 80 μl yellow micropipetting practice fluid
  • 80 μl red micropipetting practice fluid
  • tip disposal container

Reference resources
In the lab, we will use Micropipetting Practice Cards from the company miniPCR bio. Our procedure has been adapted from the following Micropipette Mastery Activity.



Troubleshooting
Before start
How to use a micropipette - We will practice pipetting small volumes of colorful liquids using micropipettes. We will work together practicing this new skill to improve our technique. Before coming to the lab watch this 5-minute video, How to Micropipette, that demonstrates 3 key pipetting skills:
  1. How to hold a micropipette
  2. How to set a micropipette to a desired volume
  3. How to use the plunger and ejector of the micropipette to measure a desired volume of a reagent

Using a pipette
To draw up liquid

Pipetting
Set volume using the volume adjustment wheel

Press a new tip onto the shaft
Press plunger TO the FIRST STOP
Critical
Dip tip into liquid
Slowly release the plunger to collect liquid into the tip.
To dispense sample
Touch tip to dispensing container
Press plunger THROUGH to the SECOND STOP
Critical
Remove tip from liquid then release plunger
Dispose of tip using the tip ejector
Activity 1
Read the volumes specified on the Micropipetting Practice Card and use your micropipette to add the correct amount of liquid to each circle.

Safety information
  • Try to be precise so that the liquid stays in the circle.
  • If you have trouble, dry the spot with a paper towel and try again.
  • If you have trouble, try using both hands. Dr. Sjogren has shaky hands and uses her non-dominant hand to stabilize the micropipette being held in her dominant hand.

Try to pick up all of the liquid without leaving any behind.

Safety information
  • Set your micropipette to the same volume of liquid that is specified for each of your drops.
  • Check to see if you can pick up all of the liquid without leaving any behind. There should be no space at the end of your pipette tip.

In the blank space at the bottom of the card, pipette Amount5 µL of water three times.

Note
  • How similar do the drops look in size?
  • If they do not look exactly the same what could be some sources of error?


On a dry place on your card place Amount4 µL water. Add Amount4 µL to the same place three more times.

Note
  • How much volume should you now have? ____________
  • Set your pipette to the volume you just calculated and try to pick up the entire drop.
  • Was there any liquid left on the card or was there space left in the tip of your pipette? ___________

Activity 2
Preparation of cards and dyes



Add Amount13.5 µL of blue dye to dot A

Add Amount17.5 µL of yellow dye to dot C

Add Amount17.5 µL of red dye to dot E

From dot A, pick up Amount2 µL and place it on dot F

From dot C, pick up Amount4.5 µL and place it on dot B

From dot E, pick up Amount3 µL and place it on dot D
Note
Now that you have set up your card, your goal is to mix these dyes with great care and precision. To mix, you will gently pipette up and down to the micropipette's first stop 2-3 times. Only pipette through to your micropipette's second stop at the end to remove all liquid from your tip to avoid making any air bubbles. Follow the guidance below:



From dot E, pick up Amount6 µL and mix it with the volume already present on dot F
From dot A, pick upAmount3.5 µL and mix this volume onto dot B

From dot C, pick up Amount5 µL and mix this volume onto dot D



Calculate how much volume (in units of microliters) should now be on each dot:



DotABCDEF
Volume (in microliters)
Notes

Set your micropipette to that volume and pick up each drop one by one and move to the open space at the bottom of your card. Each time you pick up a drop, note if liquid was left behind (code: LB), or if there is any space at the end of your pipette tip (code: S).
Note
Critical thinking Questions
1.When using a micropipette, what are some benefits to checking levels of liquid in your pipet tip?

2.When using a micropipette, did drops repeatedly get measured accurately (was there liquid left over on the card or was there empty space in the bottom of the pipette tip)? What could be some sources of error?

3.Based on your experience today, how would you rate your pipetting skill?
-Total Amateur
-Intermediate
-Pro
-Pipette Master!