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Sauer:bis-Tris SDS-PAGE, the very best: Difference between revisions
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Sauer:bis-Tris SDS-PAGE, the very best (view source)
Revision as of 08:58, 2 March 2022
, 2 March 2022→Stacking:
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===Stacking:=== | ===Stacking:=== | ||
1X bis-Tris gel buffer, acrylamide solution to 5 %, water for remainder. This mixture can be stored alongside the resolving mix and APS as a "kit". | 1X bis-Tris gel buffer, acrylamide solution to 5-6 %, water for remainder. This mixture can be stored refrigerated alongside the resolving mix and APS as a "kit". | ||
Optional: Add Bromophenol Blue to make the stacking gel blue. It really helps when loading samples and doesn't affect the performance. You can fuss over the concentration, or just add it from a stock such that the gel in the casting has a decent blue color. | Optional: Add Bromophenol Blue to make the stacking gel blue. It really helps when loading samples and doesn't affect the performance. You can fuss over the concentration, or just add it from a stock such that the gel in the casting has a decent blue color. Having the stacking blue also helps to differentiate the stocks in your kit. | ||
I aliquot 2.5 mLs stacking per gel, which allows excess. | I aliquot 2.5 mLs stacking per gel, which allows excess. | ||
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Add 10 μL TEMED per gel, mix, pour to fill the casting until slightly overflowing, insert the comb and leave the excess. Don't jam the comb all of the way in. The Bio-rad combs have small tabs that are intended as gap spacers. | Add 10 μL TEMED per gel, mix, pour to fill the casting until slightly overflowing, insert the comb and leave the excess. Don't jam the comb all of the way in. The Bio-rad combs have small tabs that are intended as gap spacers. | ||
Let polymerize. You'll notice a change in refraction as the wells polymerize up between the teeth of the comb. The polymerization typically stops before the surface and will not proceed to directly contact the comb (leaving rounded edges). Older protocols ask you to degas the solutions to promote polymerization, we found that this allows better polymerization, but accelerates the setting time to variable degrees, making it difficult to predict gel behavior. This is one reason to leave the excess solution on top. | Let the stack polymerize. You'll notice a change in refraction as the wells polymerize up between the teeth of the comb. The polymerization typically stops before the surface and will not proceed to directly contact the comb (leaving rounded edges). Older protocols ask you to degas the solutions to promote polymerization, we found that this allows better polymerization, but accelerates the setting time to variable degrees, making it difficult to predict gel behavior. This is one reason to leave the excess solution on top. | ||
Rinse with water to remove unpolymerized acrylamide. Remove comb. Be careful here, go slow. Creating too much suction or pushing the comb back in will cause the well spacers to unseat and bend. If this happens, you can carefully adjust their position with a gel loading tip once the system is set up. However, samples can then leak between lanes, so don't use it for a Western. Once assembled, carefully rinse unpolymerized acrylamide from the wells with a needle and syringe. We cut the tip off of the needle with scissors and reuse the same one over and over. | Rinse with water to remove unpolymerized acrylamide. Remove comb. Be careful here, go slow. Creating too much suction or pushing the comb back in will cause the well spacers to unseat and bend. If this happens, you can carefully adjust their position with a gel loading tip once the system is set up. However, samples can then leak between lanes, so don't use it for a Western. Once assembled, carefully rinse unpolymerized acrylamide from the wells with a needle and syringe. We cut the tip off of the needle with scissors and reuse the same one over and over. | ||
