Pipetting

Carlos Carlos Goller, Carly Sjogren

Published: 2024-02-28 DOI: 10.17504/protocols.io.n2bvj8x35gk5/v2

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

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
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Steps

Using a pipette

1.

To draw up liquid

1.1.

Set volume using the volume adjustment wheel

1.2.

Press a new tip onto the shaft

1.3.

Press plunger TO the FIRST STOP

1.4.

Dip tip into liquid

1.5.

Slowly release the plunger to collect liquid into the tip.

2.

To dispense sample

2.1.

Touch tip to dispensing container

2.2.

Press plunger THROUGH to the SECOND STOP

2.3.

Remove tip from liquid then release plunger

2.4.

Dispose of tip using the tip ejector

Activity 1

3.

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.

4.

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.

5.

In the blank space at the bottom of the card, pipette 5µL 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?

6.

On a dry place on your card place 4µL water. Add 4µL to the same place three more times.

This is what your card should look like:

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

7.

Preparation of cards and dyes

7.1.

Add 13.5µL of blue dye to dot A

7.2.

Add 17.5µL of yellow dye to dot C

7.3.

Add 17.5µL of red dye to dot E

After this step, this is what your card should look like:

7.4.

From dot A, pick up 2µL and place it on dot F

7.5.

From dot C, pick up 4.5µL and place it on dot B

7.6.

From dot E, pick up 3µL and place it on dot D

After this step, this is what your card should look like:

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:

8.

From dot E, pick up 6µL and mix it with the volume already present on dot F

9.

From dot A, pick up3.5µL and mix this volume onto dot B

10.

From dot C, pick up 5µL and mix this volume onto dot D

After this step, this is how your card should look like:

11.

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

ABCDEFG
Volume (in microliters)
Notes
12.

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 Questions1.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!

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