Mar 14, 2017
Casting of a polyacrylamide gel
- Brian Kenn Baltzar.1
- 1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
Protocol Citation: Brian Kenn Baltzar. 2017. Casting of a polyacrylamide gel. protocols.io https://dx.doi.org/10.17504/protocols.io.hchb2t6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: March 14, 2017
Last Modified: November 09, 2017
Protocol Integer ID: 5225
Keywords: polyacrylamide gel, gel, igem sdu, cast
Abstract
A protocol to cast a polyacrylamide gel.
Used in for iGEM SDU 2016
Guidelines
20% Separation gel-mix 15 mL
| 10 mL | 30% Acrylamide/bis | ||
| 3.75 mL | 1.5M Tris-HCl, pH 8.8 | ||
| 150 μL | 10% SDS | ||
| 1.03 mL | diH2O | ||
| 7.5 μL | TEMED | ADD JUST BEFORE CASTING! | |
| 75 μL | 10% APS | ADD JUST BEFORE CASTING |
4% Stacking gel-mix 15 mL
| 1.98 mL | 30% Acrylamide/bis | ||
| 3.78 mL | 0.5M Tris-HCl, pH6.8 | ||
| 150 μL | 10% SDS | ||
| 9 mL | diH2O | ||
| 15 μL | TEMED | ADD JUST BEFORE CASTING! | |
| 75 μL | 10% APS | ADD JUST BEFORE CASTING |
X% Separation gel-mix 15 mL
| 0.5 * X mL | 30% Acrylamide/bis | ||
| 3.75 mL | 1.5M Tris-HCl, pH 8.8 | ||
| 150 μL | 10% SDS | ||
| 11.03-(0.5*X) mL | diH2O | ||
| 7.5 μL | TEMED | ADD JUST BEFORE CASTING! | |
| 75 μL | 10% APS | ADD JUST BEFORE CASTING |
2x SDS-PAGE Buffer
| 3.75 mL | Tris-HCl pH 6.8, 0.5M | ||
| 24.0 mL | Glycerol | ||
| 300 μL | 1.0% Bromophenol blue eller xylene cyanol (MEGET Lidt ) | ||
| 6.0 mL | 10% SDS | ||
| -> 30 mL | diH2O |
Acrylamide/Bis 30%
| 87.60 g | Acrylamide (29.2g / 100mL ) | ||
| 2.40 g | N'N'-bis-methylene-acrylamide | ||
| -> 300 mL | diH2O | ||
| Filter and store at 4ºC | |||
1.5 M Tris-HCl, pH 8.8 (150 ml)
| 27.23 g | Tris-base (18.15 g/100 ml) | ||
| 80 mL | diH2O | ||
| Adjust to pH 8.8 with 6N HCl | |||
| -> 150 mL | diH2O | ||
| store at 4ºC |
0.5 M Tris-HCl, pH 6.8
| 6 g | Tris-base (18.15 g/100 ml) | ||
| 60 mL | diH2O | ||
| Adjust to pH 6.87 with 6N HCl | |||
| -> 100 mL | diH2O | ||
| store at 4ºC |
10% APS
| 0.10 g | Ammonium persulfate | ||
| 1 mL | diH2O |
10% SDS
| 10.00 g | SDS | ||
| 60 mL | diH2O | ||
| Dissolve with gentle stirring | |||
| -> 100 mL | diH2O | ||
Troubleshooting
Safety warnings
Remember to wear gloves as Acrylamide/Bis is very toxic.
Wash all of the hardware - make sure they are clean otherwise it may leak
Assemble the glass plates and spacers and fix them in the plastic mounting devices.
Mount the assembled glassplates on the casting stand and make sure the stand is level
Separation gel.
Add the APS to the Separation gel-mix
When the remaining gel-mix in the falcon tube has polymerized, remove the H2O from the top of the gel by tilting the whole assembly and using a napkin to remove the excess H2O.
Mix by gently inverting a few times
Add the TEMED - Cast the gel immediately after addition of TEMED up to the top.
Insert the comb onto the top of the gel.