to label molecular weight marker with P32
Christophe.Rouillon at ifremer.fr
Wed Sep 17 10:44:02 EST 2003
Thanks a lot Mike for this response!
It's actually a good idea to use the polymerase exonuclease activity.
A detailled protocol will be a good help if I plan to use this
"Michael L. Sullivan" a écrit :
> An excellent way to label ds DNA markers with P-32 is to use T4 DNA
> polymerase. It involves 3 steps.
> In the first step, the markers are briefly incubated with T4 DNA
> polymerase WITHOUT dNTPs. The exonuclease activity of the polymerase
> will chew back a bit on one strand of the markers. In the second
> step, dNTPs are added (one of which is labeled) and the strand that
> got chewed back previously gets filled back in, this time with hot
> nucleotide. Following labeling, the labeled markers can be cleaned
> up from free label with a spin column.
> I can provide a more detailed protocol if you are interested.
> >Since few weeks, I try to obtain P32 labeled molecular weight marker.
> >First, I tried to label Lambda HindIII digest and Lambda EcoRI digest
> >after dephosphorylation; signal was very low on denaturing gel.
> >Second, I tried to label linearized and dephosphorylated M13ss; it was
> >2 steps seem criticals: dephosphorylation (Phosphatase alkaline) and
> >phosphorylation (T4 Prot kinase).
> >I would like to know if somebody make routinely this type of marker.
> >Thanks a lot for all information
> Michael L. Sullivan, Ph.D
> Research Plant Molecular
> U.S. Dairy Forage Research Center
> 1925 Linden Drive West
> Madison WI, 53706
> (608) 264-5397 Phone
> (608) 264-5147 Fax
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