Home· · ·Techcomp college· · ·Application notes
Application notes
Himac Application No. 59 – Separation of Plasmid DNA
Himac Preparative Micro-Ultracentrifuge CS Series Neo-Angle Rotor S120NT
We've
shown in our himac APPLICATION it takes 3h to separate plasmid DNA by S120NT neo-angle
rotor (holding 2ml tube)1). Here we tried this separation even shorter.Then
we can prove this separation for 2 hours.
Results
Conditions
(1) Condition of ultracentrifugation
Rotor |
Speed (rpm) |
Time(h) |
Temp.(?C) |
Acc. mode |
Dec. mode |
S120NT neo-angle rotor |
120,000 |
1.5 1.75 2.0 |
20 |
"9" |
"7" |
(2) Used tube : 2PA Cone-Top tube*
*Registered trade mark of Seton
Scientific Company.
(3)
Sample preparation
We used E.coli., JM109 holding plasmid pUC19 DNA. The E.coli., JM109 was
cultured over night and crude DNA was isolated by alkaline-SDS method and other
processes and dissolved with TE buffer. We used this solution as a sample.
For each 2PA Cone-Top tube*, we provided
Sample: 1.48ml
CsCl : 1.52g
Ethydium Bromide (10mg/ml) : 50 μl
Polyethylen (10) octylphenylether (Triton X - 100) : 1 - 2 μl
We mixed these and poured the resulting mix into a tube. If this is not
enough to fill the tube, the tube should be filled with an additional filler
solution prepared in advance (that dissolve 0.93g of CsCl to 1ml of TE buffer).
Reference
1) himac APPLICATION No.46 (1995).
Product information Himac
Ultracentrifuge: