赛默飞世尔Thermo Fisher POTASSIUM A

In vitro transcription with yeast nuclear extract

buffer 500 mM Potassium Acetate, pH 7.6100 mM HEPES, pH 7.6 5 mM EDTA 25 mM MgOAc Creatine phospho kinase (sigma C-3755 165 units/mg) 2.0 mg/ml in HA + 0.1 M potassium acetate Stored in 12 microliter aliquots at -70 degrees. The enzyme is stable

Replication timing by density transfer

. (I add 0.1 mg/ml ampicillin if I'm feeling paranoid.) The doubling time of RM14-3a is ~ 2.6 hr at 23o C in glucose and 6-8 hr (depending on aeration) in acetate--longer if you're selecting for a plasmid. RM14-3a is MATa, cdc7-1ts , bar1, trp1-289

In vitro transcription with yeast nuclear extract

. Assay RNA synthesis using primer extension.     5x Acetate transcription buffer 500 mM Potassium Acetate, pH 7.6 100 mM HEPES, pH 7.6 5 mM EDTA 25 mM MgOAc Creatine phospho kinase (sigma C-3755 165 units/mg) 2.0 mg/ml