906
edits
Changes
updated cesium concentration
*GTE Buffer (50 mM Glucose, 25 mM Tris, 10 mM EDTA)
*Lysis Solution (0.2N NaOH, 1% SDS)
*Sodium Acetate (3M pH 4.8 and 3M pH 7; pH with Acetic Acid)
*Ultracentrifuge tubes (Beckman 362185)
*Water-Saturated N-butanol
#Load into two ultracentrifuge tubes then add 25 uL EtBR to each tube
#Centrifuge 60 000 RPM (250 000g) O/N in NVT90 rotor
#Withdraw the upper band with a 18g needle and reload into 0.1g/L mL CsCl for second spin
#Remove band and extract EtBr with butanol until clear
#Dialyse overnight in 2L of TE
#Precipitate with 3 vol absolute ethanol and 1/10 vol of 3M Acetate pH 7
#Resuspend to a final concentration of 5-10 mg/mL in TE