Isolation of cell nuclei for the application in the cell-free system |
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MATERIAL | |
NUCLEI BUFFER (NB): | |
COMPOSITION: | RECIPE for 50 ml: |
10 mM PIPES (pH 7.4) 10 mM KCl 2 mM MgCl2 1 mM DTT 0.1 mM PMSF 2 µg/ml Leupeptin 2 µg/ml Pepstatin 2 µg/ml Aprotinin |
10 ml of 50 mM PIPES, pH 7.4 500 µl of 1 M KCl 1.0 ml of 100 mM MgCl 50 µl of 1 M DTT 500 µl of 10 mM PMSF 100 µl of 1 mg/ml Leupeptin 100 µl of 1 mg/ml Pepstatin 100 µl of 1 mg/ml Aprotinin 37.65 ml H2O nuclease free |
NUCLEI STORAGE BUFFER (NSB): | |
COMPOSITION: | RECIPE for 1 ml: |
10 mM PIPES (pH 7.4) 5 mM EGTA 80 mM KCl 20 mM NaCl 50% Glycerol 250 mM Sucrose 1 mM DTT 0.2 mM Spermine 0.5 mM Spermidine 0.1 mM PMSF 2 µg/ml Leupeptin 2 µg/ml Pepstatin 2 µg/ml Aprotinin |
200 µl of 50 mM PIPES (pH 7.4), incl. 25 mM EGTA 80 µl of 1M KCl 20 µl of 1M NaCl 500 µl Glycerol 85 mg Sucrose 1 µl of 1M DTT 10 µl of 20 mM Spermine 10 µl of 50 mM Spermidine 10 µl of 10 mM PMSF 2 µl of 1 mg/ml Leupeptin 2 µl of 1 mg/ml Pepstatin 2 µl of 1 mg/ml Aprotinin 106 µl H2O nuclease free |