Genomic Diversity Laboratory Department of Ecology and Evolutionary Biology University of Michigan Recipes for Stock Solutions Bromophenol Blue Loading Dye (6X)* *dark blue runs faster on the gel Recipe 1: For 100 mL: 60 g glycerol into a 100-mL graduated cylinder 12 mL 0.5 M EDTA, pH 8 Add 10 mg bromophenol blue Bring up the volume to 100 mL with ddH2O Recipe 2: 100 mg Bromophenol Blue 6 g of Ficoll (Type 400) Fill up to 40 ml with dH2O Dissolve well. It takes a while. An option is to shake at 37 degrees overnight. Store at room temperature. GelRed Gel: Use dark 1.5 ml tubes. Store stock solution at dark place and room temperature. Loading Dye Mix Mix: 10 µl GelRed 1000 µl Loading Dye (6X) Vortex and centrifuge Dilute in water Mix Mix: 1 µl GelRed 100 µl ddH2O Ladder: 1 µl Ladder 1 µl Loading Dye (6X) 8 H2O TBE (Tris-borate-EDTA) (10X) 108 gm Tris Base (Trizma) 55 g boric acid 40 ml 0.5 M EDTA (pH 8.0) Add the above to 800 mL dH2O. Stir until dissolved. Adjust to 1 L with additional dH2O. Store at room temperature. 0.5 M EDTA (pH 8.0) Add: 186.1 g of disodium EDTA (Na2EDTA) 800 mL of dH2O Adjust the pH to 8.0 with NaOH (~50 ml of NaOH) Bring volume to 1 L with dH2O Stir vigorously on a magnetic stirrer* Sterilize by autoclaving Store at room temperature *The disodium salt of EDTA will not dissolve until the pH of the solution is adjusted to 8.0 by the addition of NaOH. 1 M Tris-HCl Dissolve: 121. g Tris Base (TRIZMA) 700 ml dH2O Add concentrated HCl to desired pH: pH 7.4 : 70 mL pH 8.0 : 42 mL pH 9.0 : 8 mL Fill up to volume 1 L with dH2O. Sterilize by autoclaving for 20 minutes on liquid setting. Store at room temperature. Tris-EDTA 100X (1.0 M Tris-HCl, 0.1 M EDTA, pH 8.0) 157.6 g Tris-HCL 37.22 g EDTA Bring the solution up to 1 Lt with H2O (before you must adjust the pH to 8 using NaOH) If preparing only 250 ml 39.4 g Tris-HCL 9.03 g EDTA Bring the solution up to 250 ml with H2O (before you must adjust the pH to 8 using NaOH) EDTA 0.5 M 186.1 g EDTA Bring the solution up to 1 Lt with H2O If preparing only 250 ml 46.53 g EDTA Bring the solution up to 250 ml with H2O (adjust pH to 8 with ~5 g NaOH) DTT (Dithiothreitol) 400mg/ml 0.4 g in 1 ml H2O If you will prepare the entire bottle of DTT (1 g) then you just add 2.5 ml of H2O NaCl 5 M 292.2 g in 1 LT H2O If preparing only 100 ml: 29.2 g in 100 ml H2O 5X Extraction Buffer – 40 ml Stock 2 ml Tris + EDTA (100X) 7.6 ml EDTA (0.5 M) 0.4 ml NaCl (5 M) 30 ml ddH2O SDS (Sodium dodecyl sulfate) 10% Dissolve: 10 g of SDS in 80 mL of H2O, and then add H20 to 100 mL Extraction Solution Recipe (1 RXN) Water 5X Extraction Buffer SDS 10% Prot. K 10 mg/mL DTT 400 mg/mL Total volume 520 ul 160 ul 80 ul 20 ul 20 ul 800 ul