DNA lebel

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Mon Dec 9 19:06:46 EST 1996


In article <32AB38D9.303F at bme.jhu.edu>, swalsh at bme.jhu.edu wrote:

> Hi..
> Can anyone tell me what is the best way to label a circular plasmid
> (probably supercoiled) with radioactive label.  I tried nick translation
> but the labeled mixture after passing through sephadex column and
> running a gel seems to have all oligonucleotides.  I need to keep the
> DNA as a circular plasmid. Thanks..

Depending on the radionuclide you wish to use, you could try "feeding"
your bugs some 32P pyrophosphate or inorganic phosphate and then isolate
the plasmid DNA by cesium/ethidium bromide centrifugation.

By the way, in nick translation there is DNAse I which creates nicks in a
target DNA which subsequently serves as a priming point for 5'->3'
exonucleolytic attack with concomitant 5'->3' polymerization by the
accompanying E. coli DNA polymerase I. This is why you only see
oligonucleotides of varying lengths on the gel; In essence your DNA was
"fragmented" by the DNAse and radioactive DNA was resynthesized on these
DNA fragments.

Cheers

Karl the hepB guy

-- 
Karl Fischer
tyr-2 at bones.biochem.ualberta.ca





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