Transcript Gel electrophoresis teacherVWR

Gel Electrophoresis
Teacher Instructions
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Set Up
12 groups
Mira Costa kit
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Timeline
• Prepare Gels: Up to a week in advance
• Class lab: 1-3 days
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Teach students to pipette
Load and run gels
Teach electrophoresis theory
Analyze gels
• Gels must be analyzed no later than next day after
running (stored in refrigerator overnight)
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Prepare 1X TAE Buffer
for making gels
• Measure 36ml of 50X
TAE Buffer stock
solution into the 50ml
conical TAE Buffer
measuring tube
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Prepare 1X TAE Buffer
for making gels
• Pour the 36ml of 50X
TAE Buffer stock
solution into the 2L
TAE Buffer mixing
bottle
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Prepare 1X TAE Buffer
for making gels
• Fill 2L TAE Buffer
mixing bottle to the
1800ml line with water
(tap or distilled)
• You might need to repeat
this as necessary for your
number of classes – this
bottle should prepare
enough 1X TAE Buffer
for 6 classes worth of gels
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REDO
Prepare Agar for Gels
• Measure agar powder
into the 15 ml agar
measuring tube – tap
firmly to settle to 4ml
mark
• Add measured agar
powder to agar mixing
bottle
• You’ll need to make
1 bottle per class
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Prepare Agar for Gels
• Fill each bottle to the
300ml mark with your
prepared 1X TAE
Buffer solution
– Bottles have been prechecked for calibration
• Cap tightly and shake
to mix
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Prepare Agar for Gels
• Loosen caps slightly and
place bottles in your
microwave
• Set microwave for 1-2
minutes per bottle (less is
better - you can always
add more time!)
• Allow agar solution to
come to a boil - stop
microwave immediately
once a good boil starts
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Prepare gels
• Carefully remove the
HOT bottle from the
microwave and swirl - be
careful of steam escaping
from the loose caps!
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Prepare Agar for Gels
• Check that agar has
fully dissolved or, if remelting solidified agar,
that it has all melted
back into solution
• Add water (DI or tap) as
needed to compensate
for evaporation to bring
volume back to 300ml
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Prepare Gel Casting Trays
• Allow Agar to cool until
you can just stand
holding the bottle with
your bare hand
• While Agar is cooling
assemble gel casting
trays
• 3 casting trays per class
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Pour Gels
• Carefully pour 100 ml
warm agar solution into
each assembled gel
casting trays (make sure
agar is still fully melted)
• Place 2 combs into
appropriate slots on
each tray
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How to store prepared gels
• After gels have solidified,
remove the combs
carefully lift out gel trays
– There will be some gel
‘film’ on the bottom
• Carefully slide gel out of
the tray onto a “patty pac”
paper
• 4 gels per paper
• Wipe excess gel off
casting tray and
reassemble for next pour
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How to store prepared gels
• Place papers with 4 gels in
a gel storage container
• Make sure paper edges are
free
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How to store prepared gels
• Stack second and third
(etc) layer of gels in
storage container and
place container in fridge
for up to a week
• Kit design is for 2 classes
per container
– If storing more per container, place
6 gels per layer to distribute
weight on bottom level
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Setting up prepared gels for class
• When you are ready to have
students use gels simply
carefully lift paper with gels
out of the storage container
– One layer at a time
• Carefully use spatula to lift
each gel from paper and
replace into gel tray
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Setting up prepared gels for class
• Place each gel onto flat tray
for each student group
• Try to keep gels and trays low
and level to prevent accidental
tearing of the gel
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Running gels
• Prepare 1X TAE Buffer
solution for running gels:
– Measure 16ml of 50X TAE
Buffer stock solution into
the 50ml conical TAE
Buffer measuring tube
– Pour the 16ml of 50X TAE
Buffer stock solution into
the 2L TAE Buffer mixing
bottle
– Fill 2L TAE Buffer mixing
bottle to 800ml line with
water (tap or distilled)
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Running gels
• Pour ~ 250ml of mixed
1X TAE running buffer
into each electrophoresis
box
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Running gels
• After students have loaded
their gels carefully place
each gel tray into the
electrophoresis boxes
– Keep track of which groups’
gels are where!
• Make sure the well sides of
the gels are on the BLACK
electrode side
– Back to Black, RUN to RED
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Running gels
• Top off each box as
needed with TAE to
bring level to just
cover gels
• Connect power
supplies
• Place lids on boxes
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Running gels
• Turn box power on (switch in
back)
• Use arrows:
– Adjust voltage to 120
– Adjust time to 20 min
– Can increase voltage to 150 and
decrease time to 15 if needed
• Press power
– If “lid” alarm sounds, turn off power,
adjust lid, start again
– May need to place large rubber band
around box to ensure magnetic
connection
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After Gel Run
• When the run is complete
(colors have separated) turn off
the power
• Remove the lids from the
electrophoresis boxes
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After Gel Run
• Carefully remove gel trays
from the box and depending on
time:
– Carefully slide each gel from gel
tray onto a flat tray and give back
to groups to analyze
– OR carefully slide each gel from
gel tray and place each gel on a
labeled patty pac and store back
in storage container in refrigerator
until next class meeting and then
distribute on flat trays
• WARNING - WET GELS
ARE VERY SLI PPERY! !
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Next period and so on…
• You can prepare per 2 gels for distribution while
per 1 gels are running and so on…
• Running TAE buffer is good for all classes – no
need to replace unless it gets too hot
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Clean up
• At end of day used buffer can just be flushed
down sink
– Rinse boxes and let air dry
• Used gels can be placed in general trash
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