Zhonglin.Chai at med.monash.edu.au
Mon Feb 21 23:33:02 EST 2000
>In article <20000221045925.64332.qmail at hotmail.com>, mimiliao at hotmail.com
>("mimi Liao") wrote:
>> Dear All...
>> The bacterial plasmids i transfected into mammalian cells are able to
>> replicate in mammalian cells. However i have trouble distinguishing between
>> the bacterial plasmids and the plasmids that have been replicated in the
>> mammalian cells. Does anyone know a good protocol for replication assay ?
>> Thanks in advance
>> Please reply to mimiliao at hotmail.com
>You might wish to try Dpn I digestion to distinguish input plasmid from
>those which have replicated within target cells; input plasmid will be
>digested while replicated should remain untouched.
>Sorry I don't have a reference handy but a quick scan of medline or pubmed
>should yield something.
This is probably because DpnI cut only methylated site of GATC. Plasmids
grown in dam+ strain E.coli. should be methylated, and hence digested by
DpnI, while replicated plasmids in mammalian cells are not methylated, and
resistant to the digestion.
ZhongLin Chai, PhD
Department of Pathology and Immunology
Monash University Medical School
Commercial Rd, Prahran, VIC 3181, AUSTRALIA
Telephone: (61 3) 9903 0698 (lab)
(61 3) 9903 0696 (office)
Mobile: 0413 58 1940 or International: +61 413 58 1940
Fax: (61 3) 9903 0731
email: zhonglin.chai at med.monash.edu.au
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