Performing the Experiment |
Materials for each Team:
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1
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Agarose gel 0.8% on gel tray
[per 2 or 4 student teams]
Prepared samples in labeled microfuge "reaction" tubes:
Tube #1 "lambda DNA EcoR I digest"
Tube #2 "lambda DNA Hind III digest"
Tube #3 "lambda DNA EcoR I / Hind III double digest"
Capillary micropipets (10 uL)
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5
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"Student Study Sheets" copymaster
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5
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"Student Analysis Sheets" copymaster
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200
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mL TBE running buffer (1X)
[per 2 or 4 student teams]
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1
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250 mL beaker (for distilled water & running buffer)
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5
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Pair eye goggles
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5
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Aprons/smocks
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5
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Pair protective gloves
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1
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Capillary micropipet bulb assembly
Calculators
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Common Materials:
Ward's power supply
Electro-CellTM
2 Staining trays
1 Bottle, Ward's DNA stain (100 mL per gel)
Distilled water
Spatula "gel handler"
General note: The DNA sample amounts in the labeled microfuge "reaction"
tubes are extremely small. Your teacher will demonstrate the correct procedures
needed to transfer samples from these reaction tubes to the wells on your
gel.
Loading And Running A Gel:
WEAR EYE PROTECTION FOR ALL THESE STEPS
WARNING: The power supply produces high enough voltage to cause severe
electrical shock if handled improperly. For safe operation of this unit, follow
all directions and precautions. Assure that all students are thoroughly drilled
on the procedure regarding the use of this unit, making electrical connections,
and that they are directly supervised by you. We strongly urge you to take the
time to examine all components of your electrophoresis apparatus prior to each
use. The
maintenance inspection should include all cords, plugs, Electro-Cell(TM)
apparatus, and power supply.
Precautions:
Use the same precautions as you would with any other electrical device. This
device produces high enough voltage, although low compared to general
electrophoretic standards, to cause severe electrical shock.
Designate one area of the laboratory as an "electrophoresis area" where cells
and power supplies are connected. Place the power supply near the
Electro-Cell(TM) to which it is to be connected but away from normal everyday
activities.
Do not plug Power Supply into wall receptacle until the safety cover is
positioned on the cell and all other electrical connections are properly made.
This unit uses a 3-wire grounded plug. For safety reasons it should NOT be used
with a 2-wire receptacle with a
conversion plug.
Do not operate in a damp or humid environment since any condensed moisture may
short out electrical components. Assure that the power supply, electrical cord,
and patch cords are clear from wetness or moisture.
Inspect all power cords, patch cords, banana jacks, and plugs for any defects,
such as cracked and dried-out insulation, and loose or wobbly banana jacks or
plugs.
Do not come in personal contact or allow metal or any conductive material to
come in contact with reservoir buffer or the electrophoretic cell while power
supply is on.
The power supply may continue to produce some voltage even when the power has
been turned off. To eliminate any risk associated with this event, follow all
required steps given in the procedure, when disconnecting the electrophoretic
chamber (Electro-Cell(TM)) be sure that the leads do not touch each other, come
in contact with the buffer, or otherwise create any hazardous electrical
condition.
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(1)
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On a gel, load the entire sample (10 uL) from each
microfuge tube onto a corresponding gel lane. If more
than one student team is using a gel, keep track of
which well number (from left to right) of a given
sample is placed. Be careful not to puncture the
bottom of the well. Your teacher will demonstrate the
correct procedure.
Lane 1 (Tube #1) "lambda DNA EcoR I digest"
Lane 2 (Tube #2) "lambda DNA Hind III digest"
Lane 3 ( Tube #3) "lambda DNA EcoR I / Hind III
double digest"
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(2)
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Place your loaded gel (on the gel tray) in the center
island of the cell. Orient the gel so that the wells are
closest to the BLACK (cathodic) electrode.
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(3)
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Pour approximately 200 mL of TBE running buffer
(1X) into a beaker. Use the beaker to gently pour
running buffer into one buffer compartment of the
cell. As the level reaches the gel, pour running buffer
into the other buffer compartment until it too reaches
the level of the gel. Slowly add enough running buffer
into the compartment nearest the Red (anode)
terminal until the level of buffer is approximately
2 mm ABOVE the top surface of the gel. This type of
gel is called a "submarine." DO NOT OVERFILL the buffer compartments!
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(4)
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Wipe dry the Safety Cover and place it on the
Electrophoretic Cell (Electro-Cell(TM)). Assure that the
Electro-Cell(TM) is not overfilled with buffer. The buffer
level should be approximately 2 mm ABOVE the top
surface of the gel. Wipe off any spills around the
electrophoretic apparatus and power supply before
proceeding to the next step.
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(5)
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Make sure that the power supply is unplugged and is
switched off.
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(6)
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Assure that the patch cords are completely dry,
including the female plugs, as well as the banana
jacks, on the patch cords and Electro-Cell(TM).
Connect the Red (positive) patch cord to both the Red
electrode terminal on the cell and the Red terminal on
the power supply. Follow the same procedure with
the Black (negative) patch cord.
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(7)
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Plug the power cord into a typical 120V receptacle.
Assure that the receptacle is properly wired; i.e., that
correct polarity exists.
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(8)
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Ward's 2-cell and 3-cell power supplies have a preset voltage output. If you are using Ward's 15-5340 power supply, set at 90 V. Refer to "Ward's
Agarose Electrophoresis Manual," section 2.2 for specific information regarding
voltage settings and running times.
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Teacher's Note: Refer to Ward's "Agarose Electreophoresis Manual," Section 2.2 for information regarding voltage settings and running time.
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(9)
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Turn on the power supply. The run light willilluminate signifying that
power is running to the cell.Observe the bubbles that form along the platinum
electrodes.
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(10)
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Observe the migration of the sample down the gel towards the Red (anodic)
electrode. Turn off the power when the loading dye has run halfway off the
gel.
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(11)
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Unplug the power supply.
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(12)
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For safety reasons wait approximately 10 seconds, then first disconnect
the patch cords from the power supply, and afterwards from the
Electro-Cell(TM).
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(13)
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Carefully notch one end of the gel so that future orientation will be
assured. Lift the gel tray (contain-ing the gel) from the cell and place the
gel in the staining tray. Do this by GENTLY pushing the gel off the tray using
the edge of the "gel handler."
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(14)
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WEARING PROTECTIVE GLOVES pour approximately 100 mL of DNA Stain into the
staining tray. Do NOT pour stain directly onto the gel. Cover and label.
Staining will be complete in about 3 hours.
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(15)
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Following staining, carefully decant the used stain.DO NOT allow the gel
to move up against a corner. It MUST remain flat, otherwise, it could break.
Decant the stain directly to a sink drain. Flush with water.
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(16)
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Add distilled water to the staining tray. Do NOT pour water directly onto
the gel. The tray may be gently rocked to promote destaining. Destain until
bands are distinct, with little background. This will take between
8 and 12 hours, depending upon the degree of agitation used.
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(17)
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Wash your hands thoroughly before leaving the lab.
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(18)
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Observe the gradual destaining of the gel. Destaining is completed when
DNA bands are clearly observed against an almost clear background.
Teacher's note: Use the spatula ("gel handler")
provided in the kit to remove the gel from the staining
tray. Wrap the gel in clear kitchen wrap and store
under refrigeration. (Gel bands will retain the stain
for about one month before they start to fade. Fading
is due to the oxidation of DNA since it [DNA] is not
fixed in the gel matrix.)
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(19)
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Analyze results. See your Student Analysis Sheet.
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AE Partners Collection Index
