1. Wash glass plates, alumminium plates, spacer and combs with soap, dest. water and ethanol and let them dry.
2. During the plates are drying one can prepare the needed solutions.
lower gel solution (20% AA) 22.3 ml
AA stock
4.1 ml 3 M Tris/Cl pH 8.8
5 g Sucrose
add water to a final volume of 33 ml
dissolve the Sucrose and then add
300 ul 10% SDS
300 ul Bromophenolblue (0.1%)
Degas
upper gel solution (10 % AA) 11.1 ml
AA Stock
4.1 ml 3 M Tris/Cl pH 8.8
300 ul 10% SDS
add water to a final volume of 35 ml (this will lead to a percentage slightly
lower than 10%)
Degas
3. While degassing mount the gel stack and prepare a solution of 75% Glycerol in 1x Buffer (omit the SDS)
4. Set up the pouring apparatus: Gradient mixer on a stirring
plate connected to a pump (our Rainin pump is just fine, actually the tubes
in line 2 are
the ones for pouring gels, speed is set to max. this means on the display
you should see a 48 lighting up)
The pump again
connected to the multiple gel caster equipment.
5. Add 100 ul 10% APS and 12 ul TEMED
to the lower gel solution and 20 ul APS and 12 ul TEMED to 20% solution.
Mix and pour into
the chambers of the gradient mixer, this means the lower percentage
solution goes into the cylinder close to the outlet and this cylinder is
to
be equipped with a stir bar, the higher percentage solution (blue)
goes into the other left cylinder, the valve between the cylinder is closed
at
this moment.
6. Start pouring the sandwich. To do so first check all connection
and then start the pump and the stirrer. When the first portion of the clear
gel
enters the chamber open the valve between the cylinders and make
sure that you are mixing completely, without overdoing the stirring rate.
Pump the whole fluid into the casting chamber.
7. When the last fluid is going to leave the mixer pour the 75% glycerol
solution into the mixer without introducing any air bubbles. Continue to
pump the glycerol until the The acrylamide gel has nearly completely
entered the glas sandwich, you might leave 0.5 - 1 mm under the sandwich,
no more.
Overlay the gel with 0.5 ml n-butanol, water saturated. Leave
untouched for 2 hours for polymerization to complete.
8. After polymerization is complete pour of the butanol, take
care that stuff is not healthy, and overlay with the stacking gel.
10.2 ml AA stock
3.74 ml 2 M Tris pH 6.8
0.5 ml 10 % SDS
0.5 ml APS
0.05 ml TEMED
add water to 60 ml and mix well
pour onto the top of the sandwich, make sure to fill every slot.
Insert the combs and leave the gel untouched for two hours.
9. After the gel is polymerized completely unmount the sandwich,
cut away all excessive acrylamide and pack each gel separately in saran wrap.
Store in fridge.