Gel Electrophoresis

1
Gel Electrophoresis

• Teacher Instructions
• VWR
• Set Up
• 12 groups
• Mira Costa kit

2
Timeline

• Prepare Gels Up to a week in advance
• Class lab 1-3 days
• 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)

3
Prepare 1X TAE Buffer for making gels

• Measure 36ml of 50X TAE Buffer stock solution
  into the 50ml conical TAE Buffer measuring tube

4
Prepare 1X TAE Buffer for making gels

• Pour the 36ml of 50X TAE Buffer stock solution
  into the 2L TAE Buffer mixing bottle

5
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

REDO
6
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
• Youll need to make 1 bottle per class

7
Prepare Agar for Gels

• Fill each bottle to the 300ml mark with your
  prepared 1X TAE Buffer solution
• Bottles have been pre-checked for calibration
• Cap tightly and shake to mix

8
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

9
Prepare gels

• Carefully remove the HOT bottle from the
  microwave and swirl - be careful of steam
  escaping from the loose caps!

10
Prepare Agar for Gels

• Check that agar has fully dissolved or, if
  re-melting 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

11
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

12
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

13
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

14
How to store prepared gels

• Place papers with 4 gels in a gel storage
  container
• Make sure paper edges are free

15
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

16
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

17
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

18
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)

19
Running gels

• Pour 250ml of mixed 1X TAE running buffer into
  each electrophoresis box

20
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

21
Running gels

• Top off each box as needed with TAE to bring
  level to just cover gels
• Connect power supplies
• Place lids on boxes

22
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

23
After Gel Run

• When the run is complete (colors have separated)
  turn off the power
• Remove the lids from the electrophoresis boxes

24
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 SLIPPERY!!

25
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

26
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