Q. About Gyrase

Jun Ishikawa jishikaw at ddbj.nig.ac.jp
Wed Mar 17 01:19:31 EST 1993


Dear colleague,

I'm sorry for my poor English (Japanglish?).(_O_)

I'm trying to cleave pBR322 plasmid DNA with DNA gyrase. 
Although the plasmid was cleaved at the expected site, 
the amount of DNA decreased, may be degraded, during incubation.
I'd like to ask you what is the cause of this phenomenon.
One possibility is "BRL's gyrase contains DNase (exo-, endo- or both)".
Significantly, the degradation is enhanced by the addition of ATP.
Please show me the best way or some informations.

Reaction conditions are as follows:

	50mM Tris-HCl (pH7.5)
	5mM MgCl2
	50mM KCl
	1mM DTT
	5% glycerol
	300ug/ml BSA (Fr.V)
	100ug/ml oxolinic acid (Sigma)
	1ug pBR322
	2.5units gyrase (BRL; M.luteus)

-Mix above (10ul), incubate at 25C for 1-hr.
-Add SDS & proteinase K (Merk) to a final 0.5% & 1mg/ml, respectively.
-Incubate at 37C for 30-min.
-Electrophoresis.

--
 ==============================================================
   Jun Ishikawa, Ph.D. (Mr.)     Dept. of Bioactive Molecules
                                 NATIONAL INSTITUTE of HEALTH
   jishikaw at ddbj.nig.ac.jp       1-23-1, Toyama, Shinjuku,
                                 Tokyo 162, Japan
 ==============================================================



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