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How to run an SDS-PAGE gel:

Outline:
I.
II.
III.
IV.

Casting gel
Assembling the apparatus
Loading and running your gel
Continue with your project (staining, Western Blot, etc.)

1. Casting your gel:

1. Assemble your glass plates. Here is the outline of what it is supposed to look like when you
cast your gel.

2. Install the glass plates onto a casting frame then to the casting stand as shown below:
Notice: the back glass plate and spacers are one. Therefore, you will
have one shorter glass plate in the front and a taller glass plate with
attached spacers in the back.

MAKE SURE THE BOTTOMS OF THE TWO GLASS PLATES

ARE STRAIGHT AND EVEN WITH EACH OTHER OR ELSE
IT WILL LEAK!

3.
4.
5.
6.

Make desired percentage of resolving gel WITHOUT 10% APS and TEMED
Degas your resolving gel solution with a vacuum for 10-15 minutes
After degassing, add in your 10% APS and TEMED and mix
Transfer into the glass plates to desired level then add water-butanol to smooth the surface
of the gel (see below picture)

7. Wait approximately 60 minutes for resolving gel to solidify

8. After resolving gel is set, use a Kimwipe and tilt the whole apparatus to allow butanol to pour
out and be absorbed to the wipe. Since the gel is set, your gel will be fine and will not leak
out.
9. Clean the surface of your gel with Di H2O and use the same method as above to get rid of the
water.
10. Repeat steps 3 to 5 for your stacking gel (note your stacking gel will be lower in percentage
and degassing is still necessary before the addition of 10% APS and TEMED)
11. Pour your stacking gel into the glass apparatus and insert your comb. See below:

12. Allow approximately 45 minutes to solidify

13. Once solidified, you can remove the comb GENTLY and use Di H2O to wash out the wells
before use.
2. Assembling apparatus
1. Remove your gel from the casting stand
2. Assemble your apparatus, best taught using pictures below!

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1.

2.

Make sure the shorter

glass side of your casted
gel is INSIDE the buffer
chamber!

3.
3. Pour in running buffer into buffer chamber (fill to the top)
4. Fill the outer chamber half-way with running buffer
3. Load your gel and run it an the appropriate voltage
4. Continue with your experiment involving your gel