Annex 1. Solution recipes Have to add 1mM PMSF, protease inhibitor mix and 20mM Na- butyrate right before use (calculate amounts to use) Values given in ml are from stock solutions Start Concen. Lysis Buffer Stock 1 M 5 ml - 50mM Tris-HCL (pH 8.0) 0.5 M 2 ml - 10 mM EDTA Have to add 1mM PMSF, 10% 10 ml - 1% SDS protease inhibitor mix and 17 ml + 83 ml of water = 100 ml total 20mM Na- butyrate right Start Concen. RIPA ChIP Buffer Stock before use (calculate 1 M 1 ml 10mM Tris-HCL (pH 7.5) amounts to use) – 1 M 14 ml 140 mM NaCl Values given in ml are from – 0.5 M 200 ul 1 mM EDTA stock solutions – 0.5 M 100 ul 0.5 mM EGTA – From stock 1 ml 1% Triton X - 100 – 10% 1 ml 0.1% SDS – 10% 1 ml - 0.1% Na-deoxycholate 18.3 ml + 81.7 ml of water = 100 ml total 5 ul PMSF For Lysis buffer and RIPA ChIP buffers 10 ul protease inhibitor per mL of buffer 20 ul Na-butyrate Have to add 20mM Na-butyrate, 1% SDS and 50 ug/ml proteinase K right before use (calculate amounts to use) Values given in ml are from stock solutions 100ul 10% Start Concen. Elution Buffer Stock SDS 1 M 2 ml - 20mM Tris-HCL (pH 7.5) For Elution buffer 2.5 ul 0.5 M 1 ml - 5 mM EDTA Proteinase K per mL of buffer 1 M 5 ml 50 mM NaCl – 20 ul 8 ml + 92 ml of water = 100 ml total
Annex 1. Solution recipes Have to add 1mM PMSF, protease inhibitor mix and 20mM Na- butyrate right before use (calculate amounts to use) Values given in ml are from stock solutions Start Concen. RIPA ChIP Buffer Stock 1 M 1 M 0.5 M 0.5 M From stock 10% 10% 1 ml – 10mM Tris-HCL (pH 7.5) 14 ml – 140 mM NaCl 200 ul – 1 mM EDTA 100 ul – 0.5 mM EGTA 1 ml – 1% Triton X - 100 1 ml – 0.1% SDS 1 ml - 0.1% Na-deoxycholate 18.3 ml + 81.7 ml of water = 100 ml total Have to add 1mM PMSF, protease inhibitor mix and 20mM Na- butyrate right before use (calculate amounts to use) Values given in ml are from stock solutions Start Concen. Lysis Buffer Stock 1 M 0.5 M 10% 5 ml - 50mM Tris-HCL (pH 8.0) 2 ml - 10 mM EDTA 10 ml - 1% SDS 17 ml + 83 ml of water = 100 ml total 5 ul PMSF For Lysis buffer and RIPA ChIP buffers 10 ul protease inhibitor per mL of buffer 20 ul Na-butyrate Start Concen. Elution Buffer Stock 1 M 0.5 M 1 M 2 ml - 20mM Tris-HCL (pH 7.5) 1 ml - 5 mM EDTA 5 ml – 50 mM NaCl 8 ml + 92 ml of water = 100 ml total Have to add 20mM Na-butyrate, 1% SDS and 50 ug/ml proteinase K right before use (calculate amounts to use) Values given in ml are from stock solutions 100ul 10% SDS For Elution buffer 2.5 ul Proteinase K per mL of buffer 20 ul