Berglund:SDS-PAGE

SDS-PAGE Gels
The old, longer protocol can be found here.

Separating Gel

 * 1) Mix separating gel.
 * 2) Add 50μL fresh ammonium persulfate and 16μL TEMED. (More TEMED = faster polymerization).
 * 3) Pour 4mL per gel (up to the bottom of the green bar) using P1000 pipette.
 * 4) Layer 2-methyl-1-propanol (isobutanol) or isopropanol on top.
 * 5) Leave at room temperature for >30 minutes to polymerize.
 * 6) Wash out isobutanol with distilled H2O, dry with strip of Whatman paper.

Stacking Gel

 * 1) Mix stacking gel.
 * 2) Add 50μL fresh ammonium persulfate and 12μL TEMED.
 * 3) Pour ~2mL per gel (to nearly the top of the plates) using P1000 pipette.
 * 4) Insert combs, clean spills.
 * 5) Leave at room temperature for >30 minutes to polymerize.

To use immediately

 * Put into gel box with small plate facing core of the box.
 * Lock in the plates so there are no leaks.
 * Pour 1x SDS running buffer into the core of the box so that it spills over the glass plates and into the base of the box.
 * Carefully remove combs by pulling straight up.
 * Use a pipette tip to straighten any crooked wells.

To store for later use

 * Remove plates from rack.
 * Remove comb
 * Wrap tightly with saran wrap with lightly moistened paper towel in between plates.
 * Label with name, date, and gel percentage and store at 4°C.

PLEASE don't store plates with combs, we don't have enough combs for long term storage!

Buffers
Loading buffer/dye


 * 1) Mix sample with dye, 1:4 for 5x, 1:1 for 2x. Boil at 100°C for 2 min.
 * 2) Run gel at 8milliamps until dye has reached the separating gel then at 25milliamps until dye has reached the bottom of the gel.

10X SDS Running buffer*

 * 1134g Glycine
 * 240g Tris base
 * Add to 6L of DDH2O while stirring.
 * After dissolved, bring final volume to 8L.
 * pH to 8.8, store at room temperature.

1XSDS Running Buffer*

 * Make fresh from 10X (1L of 10x into 9L of water)
 * Add 20% SDS to give final concentration of 0.1% (50ml into 10L)

*These are no longer the buffer protocols we use. Will be updated someday.