1. Volumes necessary for pouring gels of different thickness( for two 6x8cm gels) Gel Thickness Separatin Stackin 0 75mm 8.4ml 2.1ml 1.5mm 16.8ml 4.2m Always prepare with a moderate excess of gel solution 2. Calculation for x% Separating gel: Solution(C Solution(A) 2.5 H20 (7.5-x/3)ml 10% Ammonium Persulfate 50 Hl TEMED 5μl **Total Volume 10 ml *Pouring the separating gel Example of Separating Gel Preparation 12.5 %Running gel separating gel): H20 TEMED 0.02ml D(fresh) 0. 07ml Total 18ml TEMED or d was added last 1. Assemble gel sandwich according to the manufacturers instructions, or according to the usage of alternative systems. For Mini-Gel, be sure that the bottom of both gel plates and spacers are perfectly flush against a flat surface before tightening clamp assembly. A slight misalignment will result in a leak. 2. Combine solutions C and B and water in a small Erlenmeyer flask or test tube. 3. Add nium persulfate and TEMED, and mix by swirling or inverting container gently (excessive aeration will interfere with polymerization). Work rapidly at this point because polymerization will be under way 4. Carefully introduce solution into gel sandwich using a pipet. Pipet solution so that it descends along a spacer. This minimizes the possibility of air bubbles becoming trapped197 1. Volumes necessary for pouring gels of different thickness (for two 6x8cm gels) Gel Thickness Separating Stacking 0.5mm 5.6ml 1.4ml 0.75mm 8.4ml 2.1ml 1.0mm 11.2ml 2.8ml 1.5mm 16.8ml 4.2ml Always prepare with a moderate excess of gel solution. 2. Calculation for x% Separating Gel: Solution (C) x/3 ml Solution (A) 2.5 ml H2O (7.5-x/3) ml 10% Ammonium Persulfate 50 l TEMED 5 l **Total Volume 10 ml. *Pouring the Separating Gel Example of Separating Gel Preparation: 12.5% Running gel (Separating gel): A 4.5ml C 7.5ml H2O 5.9ml TEMED 0.02ml D(fresh) 0.07ml Total 18ml ⚫ TEMED or D was added last. 1. Assemble gel sandwich according to the manufacturer’s instructions, or according to the usage of alternative systems. For Mini-Gel, be sure that the bottom of both gel plates and spacers are perfectly flush against a flat surface before tightening clamp assembly. A slight misalignment will result in a leak. 2. Combine solutions C and B and water in a small Erlenmeyer flask or test tube. 3. Add ammonium persulfate and TEMED, and mix by swirling or inverting container gently (excessive aeration will interfere with polymerization). Work rapidly at this point because polymerization will be under way. 4. Carefully introduce solution into gel sandwich using a pipet. Pipet solution so that it descends along a spacer. This minimizes the possibility of air bubbles becoming trapped