smithwhi at students.msu.edu
Thu Mar 24 10:21:00 EST 1994
In Article <2mjg7v$h9e at mserv1.dl.ac.uk> "marodriguez at mvax.cbm.uam.es (marodriguez)" says:
> Dear netters,
> I'm posting this for a friend. He wants to know how to depurinate
> supercoiled DNA, and HOW to detect the amount of this depurination. The method
> he is doing now is the next:
> o Incubation of the supercoiled plasmid in Tris-HCL 0.01M (pH=5.2); NaCl
> 0.1M; Sodium citrate 0.01M, during 15-20 minutes, at 70 C.
> o Aliquots of this incubations are denaturalized by 2 hours incubation at
> room temperature, in K2HPO4-KOH 0.3M (pH=12.3).
> o Neutralization using KH2PO4-HCl 1M (pH=4.0).
> o This samples are loaded in agarose gels, stained with ethium bromide to
> detect the amount of supercoiled or relaxed plasmid.
> If you have any other method that works, please let me know about it.
Use alkaline sucrose gradients collected through a UV monitor. The
protocols are preserved in some papers by F. Studier and his coworkers from
the 1960s. The agarose gel is much to insensitive.
> Thanks in advance.
> Miguel Angel RODRIGUEZ-GABRIEL
> Centro de Biologia Molecular
> Universidad Autonoma de Madrid
> Cantoblanco. 28049 Madrid. SPAIN.
> FAX: 34-1-3974799
> e-mail: marodriguez at mvax.cbm.uam.es
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