Making a PAGE Gel

1. Collect materials that you will need. For 13 gels: 14 glass fronts, 13 white backings, 26 T spacers, 13 combs (can be any combo of 10 and 15 well i.e. 7 -15's and 6-10's)
2. Assmeble 13 gel sandwiches in a stack: white backing, T spacers, and glass front; white backing, T spacers, and glass front; white backing, T spacers, and glass front; etc.
3. Place them in the mold making sure that there is a glass one on the top so that you can see the level of liquid you are pouring in. Put an additional glass front on the top to fill out all the space. Clamp the top of the mold on.
4. Prepare the main (separating) gel solution in a graduated cylinder. Note: Make sure to add the Temed last because it causes the congealing of the mixture. I usually add in the following order:
   1. Acrylamide with bis (Fridge)
   2. Tris
   3. SDS
   4. APS (-20 in box with RNAse)
   5. Water to final volume
   6. Temed (with other small bottle chemicals in weigh boat drawer)
   After adding all the ingredients I place a piece of parafilm over the top of the graduate and invert to mix.
5. Pour the mixture directly from the graduate into anywhere in gel stack, a hole in the bottom will evenly distribute the solution. Watch the level as you pour from the front. Stop when you get to the desired height of your main gel. Pour the remaining acrylamide mixture into a beaker to solidify before disposing of. Make a note of how many gels you made and how much of the solution you used.
6. Add 200_l of gel overlay to each gel in the stack. It helps to use a pipette tip in your weak hand to keep track of where you are by placing it in the gel next to be added to. The gel overlay helps the acrylamide mixture on the top of each gel to solidify, it wont if it is in contact with the air.
7. Allow the mixture to solidify for about 30-45 minutes. The beaker of unused acrylamide mixture is a good indicator. Note: The top of this mixture wont solidify because of its contact with the air.
8. While the main gel is solidifying start preparing the stacker solution by measuring out the volume of water to be used and place it in a 50ml falcon tube (orange cap) on ice. Note: It is essential that the stacker solution be prepared on ice and kept as cold as possible to avoid solidification prior to comb insertion.
9. Add all other components of the stacker solution except the temed,, making sure to keep on ice to help avoid premature solidification.
10. Get combs ready, then add the temed, cap and invert to mix.
11. Pour stacker solution in to mold. Try to evenly distribute between gels. It should overlflow ones and distribute itself evenly to all by overflowing but try to distribute it as evenly as possible.
12. Put combs in. One goes in behind each glass front and should slip inbetween the glass and white back in the space created by the "T" spacers.
13. Allow it to congeal for 30-45 minutes. Then wrap it in moist paper towels and store in the fridge.