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[MUSIC PLAYING]

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PROFESSOR: Many
laboratory procedures,

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especially biological
and biochemical ones,

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require the precise transfer
of small quantities of liquids.

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Glass pipettes,
which are commonly

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used in chemistry
labs, are simply not

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accurate when working
with volumes that

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are less than 1 milliliter.

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The best method of transferring
small volumes of liquids

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involves the use of
an automatic pipette.

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An automatic pipette
consists of a piston

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that displaces air to
create a vacuum when

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its plunger is pressed.

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The tip of the automatic
pipette is then

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placed into the sample vial and
the plunger is slowly released.

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This action draws
liquid into the tip.

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Finally, this liquid is expelled
into the desired container

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by slowly depressing the
plunger a second time.

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As shown, a variety of
automatic pipettes exist.

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This video will demonstrate
automatic pipetting technique

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using the Pipetman.

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Using good technique
will ensure accuracy,

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which is defined
as the closeness

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of the dispensed volume
to the set volume,

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and precision, which is
defined as the reproducibility

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of individual measurements
between your samples.

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A Pipetman consists of a plunger
button, a tip ejector button,

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a volume adjustment
knob, a volume indicator,

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and a plastic shaft.

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Pipetman are available
in six different sizes.

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Five of them are depicted here.

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The number on the plunger button
indicates the maximum volume

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in microliters
that each Pipetman

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is capable of transferring.

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For example, the P20 has a
possible volume range of 0

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to 20 microliters.

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However, transferring volumes
at the low end of the range

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is not recommended because the
standard deviation is too high

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to ensure precision.

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Thus the minimum
amount of liquid

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that can be transferred in
microliters is 0.1 for the P2,

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0.5 for the P10, two for
the P20, 10 for the P100,

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50 for the P200, and 100
microliters for the P1000.

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Now that we've covered what
an automatic pipette is

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and how it works, we'll
take a detailed look

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into how the Pipetman is
used in the laboratory.

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Let's say, for
example, your procedure

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requires you to transfer
153.5 microliters of sample.

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The first step involves the
selection of the Pipetman

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with an appropriate
volume range.

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In this example, a
couple of choices exist.

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The P200 has a
recommended volume range

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of 50 to 200 microliters
while the P1000

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has a recommended range of
100 to 1,000 microliters.

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Remember the lower
end of a volume range

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always has a higher
standard deviation.

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Therefore the P200
is the better choice.

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The second step is the selection
of the proper disposable

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plastic tip.

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Three tip sizes exist--

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the large blue tips,
which fit the P1000,

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the medium yellow tips,
which fit the P200, P100,

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and the small white tips,
which fit the P10 and P2.

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Because we're using
the P200, we're

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going to need the yellow tips.

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Using the proper tip is
essential for preventing leaks

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and promoting accurate
delivery of liquids.

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After selecting the
correct Pipetman end tip,

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set the desired transfer volume.

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For the smaller
Pipetman, P2 to P200,

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the volume set on the
indicator translates exactly

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into the volume that
will be delivered.

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This P200 is set to deliver a
volume of 125.9 microliters.

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For the P1000, the volume
set on the indicator

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must be multiplied by 10 to
determine the actual volume

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that will be transferred.

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This P1000 is set to deliver
a volume of 1,259 microliters.

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To correctly adjust
the volume setting,

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turn the volume
adjustment knob to one

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third of a revolution
past the desired volume.

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Then slowly move the dial back
down to the desired setting.

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Always approach
the desired volume

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by dialing downwards to
prevent mechanical backlash

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and to ensure precise
volume transfer.

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When adjusting the
volume of a Pipetman,

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take care not to rotate
the dial past the maximum.

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You cannot transfer
volumes above the range

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of the Pipetman.

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Now it's time to
transfer your sample.

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You will need your
appropriately sized Pipetman set

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to the desired
volume, the correctly

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sized tips, a rack, the
new container, a chem wipe,

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and a waste container.

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Attach a disposable
tip to the Pipetman

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by pressing its
plastic shaft firmly

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into the tip with
a twisting motion.

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This action should
ensure an airtight seal

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and prevent leaks
from occurring.

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Always use a new sterile
tip for each sample

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and never let the tip touch
anything except sample.

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To draw a sample
into the plastic tip,

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depress the plunger
until resistance is felt.

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Hold the Pipetman vertically
and immerse it into the sample.

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After the tip is immersed,
slowly release the plunger

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to draw liquid into the tip.

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Wait a few seconds to ensure
that the full volume of sample

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is drawn into the tip.

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As a guide, wait one second
for the P2 to P200 Pipetman

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and 2 to 3 seconds
for the P1000.

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The larger the volume or
the more viscous the sample,

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the longer you should wait.

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Remove the Pipetman
from the sample

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and carefully wipe
excess liquids

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from the sides of the tip.

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To dispense the liquid
in the Pipetman,

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touch its tip to the side
wall of a new container

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and slowly depress the
plunger to the first stop.

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Wait a few seconds to
ensure complete sample flow,

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then press the plunger
to the second step

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to expel any remaining liquid.

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With the plunger
fully depressed,

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carefully remove the
tip from the container

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by sliding it up
the container wall.

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The importance of sliding
the table along the wall

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increases as the
volumes become smaller.

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Finally, allow the plunger
to return to the up position.

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If you are done working
with a particular sample,

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press the tip ejector
button to release the tip

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into the proper
waste receptacle.

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Several other
guidelines will help

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to ensure accuracy and precision
when transferring liquids.

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First, use a fresh tip for each
sample to avoid contamination.

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Second, pre-rinse each new tip.

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Most liquids leave a
film on the inside wall

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of a new tip, which affects
the actual volume that

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can be transferred.

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By pre-rinsing a tip,
these volume variances

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are eliminated.

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To pre-rinse a tip, simply
draw solution into the tip

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and expel it into
a waste container.

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Third, check the
immersion depth.

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If the immersion
depth is too deep,

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liquid may get into
the plastic shaft

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and damage the mechanics
of the Pipetman.

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If the immersion
depth is too shallow,

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air may be drawn into the tip,
which greatly affects accuracy.

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As a general rule,
immerse the weight tips

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to a depth of 1
to 2 milliliters,

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the yellow tips to a depth
of 2 to 3 milliliters,

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and the blue tips to a
depth of 2 to 4 millimeters.

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If you do find an air
bubble in the tip,

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dispense the sample
into a waste container,

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check your immersion level,
and pipette more slowly.

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If you still see an air bubble,
discard your current tip

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and use a new one.

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Fourth, release the
plunger button slowly.

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Do not let the plunger snap up.

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This violent action may force
liquid into the plastic shaft.

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Fifth, when wiping excess
liquid off the sides of the tip,

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avoid touching the tip opening
because some of the sample

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may be lost.

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Six.

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When liquid is in its tip,
never allow the Pipetman

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to tilt more than 20
degrees from vertical

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and never lay the
Pipetman on its side.

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For optimum
reproducibility, it is

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important to be consistent
with your samples.

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Specifically, use a consistent
speed and smoothness

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when pressing and
releasing the push button.

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Use consistent pressure
at the first step.

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Immerse the tip to
a consistent depth.

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And hold the petmin
at a consistent angle

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that is less than 20
degrees from vertical.

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If you are confident that
you used good technique

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and are still seeing errors
with accuracy or precision,

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the following
troubleshooting guidelines

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may fix your problem.

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To ensure that your Pipetman is
actually delivering the desired

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volume, you should
periodically calibrate it.

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To calibrate a
Pipetman, determine

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how many grams of
your liquid equals

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1 milliliter from its density.

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For example, if
water is the solvent,

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then 1 milliliter of water
equals 1 gram of water.

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Tare a small container
on an analytical balance

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and set the Pipetman at a volume
near the top of its range.

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Transfer the set
volume of liquid

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to the tared container
on the balance

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and note the mass of the liquid.

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Repeat this step five more times
and determine the mean weight

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and standard deviation
from your measurements.

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Repeat the entire
process with the Pipetman

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set at a volume near
the bottom of its range.

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If these values deviate
from what you expect,

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adjust the petmin accordingly
when you transfer liquid.

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Occasionally, the
Pipetman will begin

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to leak due to improperly
fitted tips or worn o-rings.

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To check for leaks, put a
fresh tip on the Pipetman.

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Pre-rinse the tip
and draw up liquid.

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Keep the Pipetman vertical
and wait about 20 seconds.

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If you see a droplet, then
the Pipetman is leaking.

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Two final things that decrease
the accuracy and precision

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of the Pipetman involve
pipetting samples

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below 4 or above
70 degrees Celsius

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and using the Pipetman
with acids and corrosives.

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If you absolutely must
pipette an acid or corrosive,

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then you should
disassemble the Pipetman

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immediately to inspect and
clean the piston, shaft,

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seal assemblies, and o-rings.

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To summarize, using
proper technique

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with an automatic pipette
is essential for ensuring

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accuracy and precision.

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A few key points to remember
include the following.

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Be consistent with plunger
timing and pressure as well as

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immersion time and depth.

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Use a fresh, pre-rinsed
tip for each new sample.

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Check for air bubbles and
leaks during each transfer.

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And calibrate the automatic
pipette periodically.

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In addition, take care
not to drop the expensive

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and easily damaged Pipetman.

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Do not allow the tip to
touch anything except sample.

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Do not introduce liquid
into the plastic shaft

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by letting the plunger
snap up or by laying

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the Pipetman on its side.

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Finally, avoid
using the Pipetman

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under extreme
temperature conditions

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or with acids and corrosives.

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When used with
consistency and care,

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an automatic pipette
is an excellent means

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of transferring microliter
quantities of liquid.

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[MUSIC PLAYING]