Note 1: If you are preparing your own sodium chloride stock solution rather than buying it in solution, you will have trouble getting it to dissolve up to 5M. In that case, use 4M sodium chloride and 49.75 uL of it, and add 0 uL of water so that the final volume is the same.

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Note 2: Right before gelation, 2 ul each of 10% TEMED and 10% APS will be added, bringing the final volume up to 100 ul (see below).

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Step 2. Prepare gelation chamber: Obtain two glass slides (~ 3"x1"), two coverslips (~ 22mmx22mm), and some parafilm. Wrap one of the slides in parafilm such that one face of the slide has a smooth flat surface one parafilm layer thick. (The other face of the slide will have the folded edges of the parafilm on it.) Press the parafilm on the flat side to ensure there is no gap between the parafilm and the slide. Place a coverslip at each end of the parafilm-wrapped slide on its smooth face. Place this parafilm-wrapped slide with coverslips into a petri dish or any container with a lid. Set the remaining glass slide aside.

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Step 3. Prepare 10% (v/v) TEMED and 10% (w/v) APS. 10% TEMED can be made by simply diluting pure TEMED tenfold in water. 10% APS can be made by measuring out some APS, finding its weight in milligrams, multiplying the weight by 9.5, and then adding that many microliters of water. Eg for 30 mg of APS, add 285 ul of water. (This assumes that 10% (w/w) APS is ~ 5% (v/w), which is approximately correct.)

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Step 4. Prepare Gelation Solution: Mix the following 4 solutions on ice: Monomer solution, 10% TEMED (accelerator), 10% APS (initiator solution). (APS initiator solution needs to be added last to prevent premature gelation). Solutions should immediately be vortexed or pipetted up and down after mixing to ensure full mixing and the gel should then immediately be cast to prevent premature gelation. For 50 µL gelling solution, mix the following: a) Monomer solution (48µl) b) Accelerator solution (1µl): 10% TEMED (TEMED stock solution at 10%, final concentration 0.2% (w/w). (Accelerates radical generation by APS). c) Initiator solution (1µl): APS (APS stock at 10%, final concentration 0.2% (w/w)). (This initiates the gelling process. This needs to be added last).

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Step 5. Polymerize the Gel: Pipet 10 ul of gelation solution onto the flat surface of the parafilm-wrapped slide between the coverslips and place the other glass slide on top so that it rests on the coverslips, leaving a small space between the two slides where the gel will polymerize. Put a wet kim-wipe or tissue in the container to keep the air in the container hydrated. Put the lid on the container and place it in a 37C incubator for 1 hour (or 1.5 hours at room temperature) to polymerize. (Note that the most ideal condition for polymerization is oxygen-free because oxygen exposure inibits the polymerization process. However, only the edges of your gel will be affected by air exposure, and the middle of the gel will polymerize well.)

 

Step 6. Expand the Gel: Pry apart the two slides using foreceps, a razor blade, or some other thin edge. Put ~ 0.5 cm of deionized water in a petri dish or other container. Wet the paintbrush with water, use it to gently peel the gel off the slide and transfer it into the container with water. Let it sit in the water for ~10 mins, then remove the water and replace it with fresh water. Repeat this wash step 2-3 more times. The gel will expand ~4-fold. Be careful not to suck the gel up into a pipettor while changing the water!

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Step 7. Play around with the gel to see how to pick it up and transfer it to other containers. The gels are very delicate when expanded, so this takes some finesse. Try picking up the gel by pushing it onto a coverslip using a paintbrush. If the gel is too large for this, cut it to make it smaller.