200-1000 2 7 8 Micropipette Tutorial From the Science Dept at SHS.

200-

10002 7

8Micropipette

TutorialFrom the Science Dept

at SHS

200-

1000

μL

2 7

8This is a Micropipette it is used to accurately transfer small quantities of liquid.

On this model the desired volume can be adjusted within the range of microliters (μL).

1 μL = 1/1000 of a mL (milliliter)1 mL = 1/1000 of a L (Liter)So…1 μL = 1/1,000,000 of a Liter

200

μL

Some Micropipettes have preset volumes and cannot be adjusted.This pipette will only deliver 200μL.

Out Micropipettes can be adjusted.

Several different ranges

0.5 to 10 uL2 to 20 uL20 to 200 uL200 to 1000 uL

NEVER set the micropipette above or below its intended volume range

How does it work?It works much like a syringe that would deliver an injection.

Inside there is a spring loaded piston that moves up and down.

They are veryFRAGILE, EXPENSIVE,& COOL!

Be careful!!

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8

The plunger has two stops: - at the First Stop the piston moves through the volume that is set on the pipette. (In this case 278μL)

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82

7

8

278μL

The second stop is extra travel used for certain circumstances like evacuating the tip, or drawing up more than the volume indicated on the pipette

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82

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278μL

- Extra volume (? μL)

- Find all three positions.

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8

200-

1000

2 7

8

Set the correct volume on your micropipette by turning the adjustment knob which is typically the plunger button or a dial on the top portion.

This pipette’s range is 200-1000 μL and is set to deliver 278μL

Setting Volume

20-1

00

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8

Settings can vary between models and manufacturer s.

This pipette’s range is 20-100 μL and is set to deliver 27.8μLHowever you might see the following readings for this exact setting on different pipettes:

Setting Volume

2 7 8

2 7 . 8

2 7 8

The first two make the decimal places obvious,

while the last one implies the tenths place if you know the range.

1.00

-10.

00

2 7

8

Settings can vary between models and manufacturer s.

This pipette’s range is 1-10 μL and is set to deliver 2.78μLHowever you might see the following readings for this exact setting on different pipettes:

Setting Volume

2 7 8

2 .7 8

2 7 8

Some companies will change the color of the plunger button.

Attaching tipBe sure to choose the proper size tip. Instructors will set these up for you.

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Attaching tip

Press the pipette into the tip firmly to create an airtight seal.

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Obtaining a Sample

STEP ONE – press plunger to first stop and hold.HOLD

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Obtaining a Sample

STEP TWO – Insert tip into sample only far enough to ensure it stays submerged but not to the bottom where it will get blocked

HOLD

KEEP the pipette VERTICAL at all times

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Obtaining a SampleSTEP THREE – Allow plunger

to return to the home position SLOWLY so you don’t

draw in air bubbles, or splash

sample up into tip or the pipette itself.


Obtaining a Sample

Removed tip from sample before the plunger was all the way home

Likely allowed the plunger to move to home too quickly.

CORRECT

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Delivering the Sample

STEP FOUR – insert tip into the area you wish to deliver your sample. (in this case a gel for DNA fingerprinting)


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STEP FIVE – depress the plunger slowly to the first stop, then continue to the second stop, this will evacuate the entire contents of the tip. And HOLD

HOLD


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STEP SIX – While still holding the plunger at the second stop. Withdraw the tip from the well.

HOLD


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STEP SEVEN – Allow the plunger to return home.


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Discarding the Tip

STEP EIGHT – Place tip into the opening of the waste container, then depress the tip ejector.

WASTE

Tip Ejector

Be sure to use a new tip each time.


Things to AVOID !!

278

Never use a pipette in anything but a vertical orientation.

Things to AVOID !!

27

8

Never use a pipette without a tip.

HAPPY PIPETTING

2006-2007

BiotechnologyGel Electrophoresis

Comparing cut up DNA• How do we compare DNA from different

organisms?– cut up DNA into fragment• Restriction enzymes

– separate fragments by size• How do we separate DNA fragments?– run it through a gelatin – gel electrophoresis

• How does a gel work?

Gel electrophoresis• A method of separating DNA

in a gelatin-like material using an electrical field– DNA is negatively charged– when it’s in an electrical field it

moves toward the positive side

+–

DNA

“swimming through Jello”

• DNA moves in an electrical field…– so how does that help you compare DNA

fragments?• size of DNA fragment affects how far it travels– small pieces travel farther– large pieces travel slower & lag behind

Gel electrophoresis

+–

DNA

“swimming through Jello”

How does it work• The gel is made from agarose which is from seaweed• Holes (or wells) are made at one end• The DNA samples are loaded with a micropipette in

the wells• The negatively charged DNA moves toward the

positive charge electrode: “run to red”

Running a gel

1 2

cut DNA with restriction enzymes

fragments of DNAseparate out based on size

3

Stain DNA– ethidium bromide

binds to DNA– fluoresces under UV

light– HS uses a different

stain

DNA fingerprint• Why is each person’s DNA pattern different?– sections of “junk” DNA• doesn’t code for proteins• made up of repeated patterns

– CAT, GCC, and others– each person may have different number of repeats

• many sites on our 23 chromosomes with different repeat patterns

GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTTCGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA

GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTTCGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA

Allele 1


repeats

DNA patterns for DNA fingerprintscut sitescut sites

GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTTCGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA

1 2 3

DNA – +allele 1

Cut the DNA

Person 1


Differences between peoplecut sitescut sites

DNA – +person 1

Person 2: more repeats

GCTTGTAACGGCCTCATCATCATCATCATCATCCGGCCTACGCTTCGAACATTGCCGGAGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA

DNA fingerprint

person 2

1 2 3

Uses: Evolutionary relationships• Comparing DNA samples from different

organisms to measure evolutionary relationships

–

+

DNA

1 32 4 5 1 2 3 4 5

turtle snake rat squirrel fruitfly

Uses: Medical diagnostic• Comparing normal allele to disease allele

chromosome with disease-causing allele 2

chromosomewith normal allele 1 –

+

allele 1allele 2

DNA

Example: test for Huntington’s disease

Uses: Forensics• Comparing DNA sample from crime scene

with suspects & victim

–

+

S1

DNA

S2 S3 V

suspects crime scene sample

DNA fingerprints• Comparing blood

samples on defendant’s clothing to determine if it belongs to victim– DNA fingerprinting

Electrophoresis use in forensics• Evidence from murder trial– Do you think suspect is guilty?

“standard”

blood sample 3 from crime scene

“standard”



blood sample from victim 2

blood sample from victim 1

blood sample from suspect OJ Simpson

N Brown

R Goldman

Uses: Paternity • Who’s the father?

+

DNA

childMom F1 F2–

200-1000 2 7 8 Micropipette Tutorial From the Science Dept at SHS.

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