DNA Fingerprinting
TOOLS AND TECHNIQUES
Intoducing the microLiter!
 A TINY amount…a millionth of a Liter
 Very difficult to measure because it is SOOO small
 Incredibly accurate when measured correctly
 Tools that measure these amounts are therefore INCREDIBLY
expensive…be VERY careful with them!!!
 If a milliliter (mL) is 1/1000 of a Liter…
 A microliter (μl) is 1/1000 of a millileter (mL)
 The greek letter “mu” is used to represent micro (μ)
 Let’s PRACTICE 
 Work through the second page of the packet
“DNA Fingerprinting – Practice Worksheet”
The TOOLS
 DNA Fingerprinting makes use of tools that can work
with incredibly SMALL volumes
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We only need a very small sample of DNA
We can run hundreds or thousands of samples at once in small
spaces
We can be INCREDIBLY accurate and precise
We can use robotic assistance to run the full analysis
The Micropipette
 Are like a normal pipette…but
better 
 They can hold incredibly small
amounts of liquid with
incredibly high accuracy and
precision
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This makes them VERY
expensive!!!
 Two types that we will use:
 P20: Can hold up to 20 μl
 P200: Can hold up to 200 μl
Draw and label a picture to refer back to:
Setting the Micropipette
 Each micropipette has a dial for its particular volume
maximum
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NEVER set the dial above the MAXIMUM listed, this will
damage the micropipette
For a p20…NEVER set above 20
 The three numbers represent the three place values
 Starting with the largest POSSIBLE place value
 Let’s PRACTICE with the second half of practice
worksheet
Techniques for Using Tips
 NEVER TILT THE PIPETTE!!!
 NEVER USE A NAKED PIPETTE!!!
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We use tips to hold liquid, not the pipette itself
Liquids can actually damage (very severely) the internal parts
 To pick up a tip, push the pipette into the tip holder
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ALWAYS keep the tip box COVERED
This keeps out contaminants
 After using ANY liquid, we will switch tips
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This will also help minimize the risk of contamination through
mixing of samples
 To eject a tip, push down on the SIDE button
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This is attached to the metal rod that will push down on the tip
Always eject tips into a WASTE BIN
Techniques for using a Micropipette
 Push the PLUNGER to the FIRST Stop
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DO NOT GO FURTHER THAN THE FIRST STOP
We must create a vacuum in order to pick up liquid
 While holding it down, insert the tip into your liquid
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DO NOT LET GO UNTIL YOU ARE IN LIQUID
This will pick up air, not the liquid
 Release the plunger to pick up the liquid
 To eject, move the tip into the desired container
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Hold the tip against the inside surface of the container you wish to
eject into
Push the PLUNGER to the SECOND Stop to eject ALL liquid
 Let’s practice! 
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Get a pipette each and practice picking up and ejecting a tip, and
pushing the plunger to the FIRST and SECOND stop
Using the microcentrifuge
 NEVER LOAD IT SIDEWAYS
 Like a washing/drying machine, it will spin out of control and
potentially break
 ALWAYS balance the load
 In a 6-holder, this means you can load…
NOT One
 Two
 Three
 Four
 NOT Five
 Six

Balance the epitubes in the rotor
Let’s practice!!!
 Do activity One and Two on the second instructional
handout
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Answer the first two questions by converting
Answer questions 3 and 4: the “Lab Observations”
 We will discuss and answer remaining questions
after returning from break 
QoD: Final DNA Fingerprinting Day
 Summarize in a few brief sentences what we have
done in this multi-day lab. Use and define the
following terms:
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DNA (mother, baby, suspected father)
Restriction Enzyme (HindIII)
Agar gel
Comb and wells
Loading dye (indigo)
Gel chamber and buffer
Gel electrophoresis
DNA Fingerprinting
DAY ONE:
CUTTING DNA WITH
RESTRICTION ENZYME
DAY TWO:
CASTING GEL
ADD LOADING DYE
LOADING DNA
RUNNING ELECTROPHORESIS
DNA Fingerprinting: Day One
 We use a restriction enzyme to cut open our DNA
samples
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Restriction enzymes are derived from bacteria, and named
after them (EcoRI is from E. coli bacteria)
They work like crooked scissors: cut sideways
 DNA pieces are run through gel electrophoresis, and
spread out based on size.
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DNA starts at the negative end and runs towards the positive.
Negative: Black
Positive: Red
 We can use the lines of DNA to identify individuals
by their DNA fingerprint.
DNA Fingerprinting: Day Two
 We added a loading dye to run ahead of and behind
the DNA. Since you can’t see DNA in gel
electrophoresis, this helps show how far it has run.
 We loaded the DNA into gel wells and ran through
the chamber for 30-50 minutes
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Buffer solution helps conduct electricity
DNA is pulled from BLACK (negative) to RED (positive) end
 We are then left with lines of DNA of specific sizes,
measured in kbp (Kilobase pairs)
The DNA
fragments are
pushed with
electricity to
spread them
out. This creates
a DNA
fingerprint