Apurinic sites?

marodriguez marodriguez at mvax.cbm.uam.es
Mon Mar 21 01:52:47 EST 1994


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.

     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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