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