Transcript Gel electrophoresis

4.4 Using Gel Electrophoresis to Study Gene Molecules
Components of Gel Electrophoresis
• Powdered agarose
• Boiling buffer solution
Agarose Gel Concentrates
Most commonly used when separating pieces of DNA
no smaller than 50 bp and no larger than 25,000 bp
Gel Stains
The gel is “run” until molecules of different sizes
are thought to have completely separated.
Agarose Gel Tray. Gel trays differ depending on
the manufacturer. Each has some method of sealing
the ends so that liquid agarose can mold into a gel.
Some gel trays, such as those made by Owl
Separation Systems, make a seal with the box, so
casting a gel is simple. Other trays require masking
tape on the ends to make a mold. Still others, like
the one shown here, have gates that screw into
position: up for pouring the gel and down for running
the gel.
Molecules in a Gel Box. If negatively
charged molecules are loaded into the
wells and run on the gel, the smaller
ones run faster and farther than the
larger ones toward the positive
electrode. This is because smaller
molecules pass more easily through the
tiny spaces of the gel network.
Vocabulary
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Gel electrophoresis – a process that uses electricity to separate charged molecules,
such as DNA fragments, RNA, and proteins, on a gel slab
Agarose – a carbohydrate from seaweed that is widely used as a medium for
horizontal gel electrophoresis
Polyacrylamide – a polymer used as a gel material in vertical electrophoresis; used
to separate smaller molecules, like proteins and very small pieces of DNA and RNA
Ethidium bromide – a DNA stain (indicator); glows orange when it is mixed with
DNA and exposed to UV light; abbreviated EtBr
Methylene blue – a staining dye/indicator that interacts with nucleic acid molecules
and proteins, turning them to a very dark blue color
High through-put screening – the process of examining hundreds or thousands of
samples for a particular activity
4.4 Review Questions
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4.
Agarose gels can be used to study what size of DNA fragments?
If agarose gel material is labeled 1%, what does the 1% refer to?
What causes molecules to be separated on an agarose gel?
Name two common DNA stains that are used to visualize DNA on
agarose gels.
Hold
micropipet
and
epitubes at
eye level
Micropipet Use
1
2
3
4
Twist dial to desired volume
Add disposable pipet tip
Press plunger to first stop
Insert pipet tip into solution to be
transferred
5 Slowly release plunger to retrieve liquid
6 Move pipet tip into desired tube
7 Press plunger past first stop to second stop
to transfer liquid, keep the plunger down
as you remove it from the tube.
8. Eject tip
Micropipetting
technique A
technique B
Close the tips!
Technique
C
Billiard style
Micropipet
tip should
be ABOVE
the well
NOT IN
IT!!!!
Micropipet
tip punched
right
through the
gel
See dye
under the
wells
NICE!