[Music]
Welcome to Addgene's Intro to the Lab Bench!
I'm Diego,
and I'm here to talk about one of the most important tools
you'll encounter in your lab work...
a pipette.
[Intro Music playing]
This small object is an essential tool
for nearly everyone in molecular biology.
Using a pipette, you’ll be able to accurately dispense
small amounts of liquid,
amounts between point one microliters, and 1 milliliter,
far too small to eyeball accurately.
Your lab work needs to be precise,
and any changes, even very small ones,
can negatively impact your experiment results.
This video will teach you proper pipetting technique,
and how to select the proper pipette and tip for the job.
So, let’s get into it!
First, gather up your materials.
You'll need...
Pipette tips...
A waster container...
Containers to hold your measured liquid,
like a microcentrifuge tube, a bottle, or something else...
Liquid to pipette.
For this demonstration, we’ll just be using water.
Something to label your containers...
[marker squeaking]
And of course.,
a pipette!
What kind of pipette you will need
depends on your experiment.
This video will only be covering single channel pipettes.
If you’re looking to improve
your multi-channel pipetting technique,
check out Addgene’s protocol video
on multi-channel pipettes.
Back to single channel,
you’ll want to select the pipette based on the amount
of liquid you’ll be pipetting.
There are five standard sizes to choose from:
P2...
P10...
P20...
P100...
P200...
and P1000.
Have your pipette?
Great!
That means we it's time to talk about
the anatomy of a pipette.
[Music]
Here's a P1000 pipette.
From the front, you can see the plunger at the top.
The plunger is used to both draw liquid into
and expel it from the pipette tip.
Next, in the middle, you can see the volume display.
This shows the set volume amount
that will be drawn up into the tip.
We’ll show you how to read this display a little later.
Finally, we have the tip ejector ring.
This pushes the pipette tip off the pipette,
allowing you to quickly dispose of used tips.
We’ll also explain why that’s important in a bit.
Turning the pipette on its side,
you’ll see the volume adjustment ring.
This is what you’ll use to set the volume
of liquid the tip will hold.
Next you’ll see the tip ejector button,
and further down the tip ejector itself.
This is the mechanism that physically pushes the tip
away from the pipette, for those easy tip disposals.
Feeling more comfortable with the pipette?
Great!
Let’s talk about setting and reading the volume
on the pipette.
Setting the volume is the same across all standard pipettes.
Just rotate the ring below the plunger to increase
or decrease the volume.
But,
reading the display is slightly different
across different pipette sizes.
For a P1000 pipette,
the first red digit is thousands of microliters,
the middle is hundreds,
and the third is tens.
So, this is 1,000 microliters.
While this would be 650 microliters.
For the P200,
he first digit is hundreds,
the second is tens,
and the third ones.
So, one-zero-zero
from top to bottom, would be 100 microliters,
while zero-nine-five
from top to bottom would be 95 microliters.
The P20,
P10,
and P2 all follow similar patterns.
Keep in mind that this dictates what tips you’ll be using.
Different tips hold different volumes of liquid.
Check the information on the box
find out the volume range for each tip.
Now that you’re familiar with the pipette and tips,
let’s talk about using the pipette.
First, set the desired volume of the liquid
using the adjustment ring.
It’s often standard practice to go above,
then roll the dial back down the desired volume.
On some pipettes,
this can be achieved by rotating the dials
behind the numerical display.
To start pipetting, you’ll want to hold the pipette like this,
with your index finger below the notch,
and your thumb on the plunger like so.
Next, select the pipette tip designed to fit your pipette.
To load the tip,
open the tip box,
and push the end of the pipette onto the tip.
Before you move on,
ensure the tip is properly set in the pipette.
Do not touch the tips with your fingers.
Doing so could contaminate the tips,
or puncture the tip of your glove.
Now, gently push down the plunger.
You may notice several “stops,”
where you feel resistance while pressing down.
Stop at that first “stop,”
when you first start to feel that resistance.
This expels the exact amount of air from the tip
equal to the amount of liquid you previously set.
Keep the plunger held down,
and gently lower the tip into the liquid.
[liquid pouring]
Do not submerge the entire pipette.
Only the tip should ever be submerged into the liquid.
Once the tip is submerged,
slowly release the plunger,
allowing it to go back to its resting position.
Wait until the liquid has finished flowing into the pipette tip.
Keep in mind, the more viscous the liquid,
the lower the flow rate into the pipette tip.
Here's a pro tip!
If you’re pipetting from a relatively large container
holding a small amount of liquid,
tilt the container so it’s easier to reach the liquid.
Once the liquid has finished drawing into the pipette tip,
lift the pipette so that the tip exits the liquid.
When you’re lifting the pipette tip from the liquid,
no liquid should drip from the tip.
If it does, this could indicate
the tip is not properly seated on the pipette.
Now,
place the pipette tip into the container you're using
to hold the measured liquid.
This is your microcentrifuge tube, conical tube, or plate.
If the tube already has liquid,
insert the tip into the liquid.
Now, slowly press down the plunger to the first stop.
Wait,
then press down all the way to the second stop.
This ensures that you’ll expel any excess liquid from the tip.
If this is a new container,
Place the tip lightly against the side of the tube.
Capillary action between the side of the tube and the liquid
Will help draw all of the liquid out of the tip.
Now, lift the pipette tip completely out of the liquid,
and relase the plunger,
being careful to not touch the sides of the container
with the pipette.
Discard the pipette tip by holding the tip over
your dedicated waste container
and pressing the tip ejector button.
If you’re pipetting biohazardous material,
make sure to discard the tip into a biohazardous waste container.
Remember, never reuse pipette tips.
Reusing tips could lead to contamination
or inaccurate liquid amounts.
Always use a fresh tip when you’re pipetting liquid.
If you're struggling with hand steadiness
or accuracy,
try putting two fingers on the pipette for support.
You can also rest the elbow of your pipetting arm
on the bench for added stability.
Congratulations! You just successfully used a pipette!
If you had any trouble, don’t worry.
Trust me, you’ll get plenty of practice in the lab.
As mentioned before, if you’re pipetting the same amount
of liquid into different tubes or into the wells of a microplate,
you may want to use a multichannel pipette.
Check out Addgene’s video on mulitchannel pipettes
to learn more.
Thanks for watching another Addgene Intro to the Lab Bench video.
Make sure you leave a comment down below,
tell us how you like the video,
or let us know what topic you’d most like to see us cover.
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