DSI Basics of Pipetting (1)

7/30/2019 DSI Basics of Pipetting (1)

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Basics of Pipetting

Used to Transfer Liquids Accurately and Precisely

DSI: Dolphin Science Investigators

Midwest Research Institute

Biotech Fridays


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What is a pipette? A pipette (or pipet) is a laboratory instrument used to

transport a measured volume of liquid.

Pipettes are commonly used in chemistry and

molecular biology research as well as medical

testing.

Pipettes can come in several different designs for

various purposes as well as both manual and

electronic types.

A pipette works by creating a vacuum above the

liquid-handling chamber (tip) and selectively

releasing the vacuum to draw and dispense liquid.


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Types of Pipettes

Air Displacement Pipettes:recommended for aqueoussolutions and generallaboratory work.

The piston is part of the pipetteand moves a cushion of airbetween itself and the sample inorder to aspirate (draw up) anddispense the liquid.

The volume of air displaced isequivalent to the volume of liquidaspirated.


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Types of Pipettes

Positive DisplacementPipettes: better suitedfor use with viscous

(thick) and corrosive(damaging) liquids The piston is part of the

disposable pipette tip andforms a seal in the pipette

tip so that the sample is indirect contact with thepiston (no air cushon).

Performs in a mannersimilar to a syringe.


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We will use air displacement pipets.


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Single Channel vs. Multi-Channel

Single channel pipettes are for pipetting from

one sample container at a time

Multi-channel pipettes are for high throughput

applications and pipette from multiple sample

wells at once.


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Bulb Pipettes Bulb pipettes are a less

advanced option for pipetting

but may be all that is available.

The plastic bulb is attached to

the top of a disposable

serological pipet. Aspiration is

accomplished by squeezing the

bulb, then placing the pipet into

the liquid to aspirate and slowly

releasing pressure on the bulb

until the correct volume has

been drawn up.


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Bulb Pipetting

4.49 mL reading 4.88 mL reading 5.00 mL reading

Reading the Volume

Determine the volume of solution in a pipet by reading the bottom

of the meniscus at eye level. Record the volume using all certain

digits and one uncertain digit. Certain digits are obtained from

calibration marks. Uncertain digits (the last digit in the number) are

estimated between calibration marks. Some examples are shown

below:

meniscus

This method is not very accurate since the volume aspirated is controlled

visually by the user instead of a mechanical set point. It should only be used

when highly accurate volumes are not required.


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Variable vs. Fixed Volume

Variable volume pipettes are the most common

found in laboratories. This pipette can cover a

specified range of volumes and is set by turning

the volume adjustment knob. Fixed volume pipettes can be used when the

same volume is being aspirated and dispensed

many times. The fixed volume pipette will only

aspirate the specified volume but usually has abetter accuracy and precision for that volume

than a variable volume pipette might.


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Manual vs. Electronic

In manual pipettes the piston action is

accomplished by the user manually depressingand releasing the plunger with their thumb.

Electronic pipettes do the work for you the

piston action is controlled by the pipette itselfthe user pushes a button to aspirate and

dispense the liquid. Electronic pipettes reduce

the force needed by the users thumb and are

helpful when repetitive pipetting is required.Electronic pipettes often have multiple options

included such as mixing and multi-dispensing

of the sample.


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Examples of Manual and Electronic

Manual

single

channel

Manual

multi-

channel

Electronic

single

channel

Electronic

multi-

channel


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Parts of a pipette


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Our DSI Pipets

The pipetting devices we will use measure

microliters (abbreviated mL).

Sometimes, for convenience, it is typed ul,

but this is not technically correct.

A microliter is 1/1000th of a milliliter, or

1/1,000,000 of a liter.

In other words

SMALL!

http://www.earthzine.org/2009/01/06/c-more-researchers-in-

hawaii-probe-marine-microbes-train-a-new-cadre-of-scientists/
http://www.earthzine.org/wp-content/uploads/2008/12/doran-2.jpg


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You have to look very carefully to see

what you are pipetting.

http://www.yale.edu/caccone/ecosave/images/pipetting.jpg

It could be

even less than

this!


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Accuracy and Precision

Accuracy is when multiple sample deliveries at a set volume

average to the expected volume.

Precision is when multiple sample deliveries at a set volume are

close to each other.

These specifications are independent of one another.

The goal is to be both accurate and precise in your sampledeliveries. With proper technique, conditions, and care of the

pipettes this can be achieved.

low accuracy

high precisionhigh accuracy

low precision

high accuracy

high precision


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Environmental Factors Affecting

Pipetting Air pressure

Temperature of air, pipette, tips, andliquids

Evaporation rates

Surface tension of liquids Relative Humidity


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Ways to optimize pipette performance

Choose the right pipette for the job. Check for leaks or any other pipette malfunctions. If the

pipette does not appear to be functioning properly ensure

that the tip is properly fitted on the pipette and that it is set

to the correct volume. If it is still not functioning remove it

from use and notify the lab manager to have it serviced.

Choose the correct pipette tip

Correct size

Correct style Have pipettes calibrated and serviced regularly.

Read the Manufacturers Manual to understand the pipette

being used.


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Tips to minimize pipetting errors Choose the correct pipette volume for best accuracy

and precision the volume being pipetted should be within

35% - 100% of the pipettes range.

Allow all liquids and equipment to equilibrate to ambient

temperature before beginning work.

Pre-rinse the pipette tip by aspirating and dispensing the

sample liquid at least 3 times before aspirating a samplefor delivery.

Immerse the tip vertically into the sample liquid well clear

of the container walls and bottom and at a depth of

approximately 2 5mm below the meniscus.


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Tips to minimize pipetting errors

Aspirate using a consistent speed, rhythm, and plunger

pressure. Hold the tip in the sample for 1 second after aspiration

and withdraw the tip slowly and smoothly without

touching the sides of the container.

To dispense touch the pipette tip to the sidewall of thecontainer where sample is to be delivered just above the

liquid the sample is being dispensed into. Use consistent

speed, rhythm, and plunger pressure to dispense.

Put the pipette in its stand between pipetting cycles toavoid warming the pipette in your hand this can affect

the volume of liquid dispensed.


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References

www.rainin.com

www.artel-usa.com

http://www.gilson.com Practical Pipette Calibration Presented

by Larry Newman of Anmar Metrology,

Inc.
http://www.rainin.com/http://www.artel-usa.com/http://www.gilson.com/http://www.gilson.com/http://www.artel-usa.com/http://www.artel-usa.com/http://www.artel-usa.com/http://www.rainin.com/

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DSI Basics of Pipetting (1)