PEI tlc method

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Sun May 9 18:30:05 EST 1993


>Klaus, I saw your note on the network.  I would like to get your PEI tlc method
>for determining incorporation of kinased oligos.  Thanks
>_______________________________________________________________________________
>Dean R. Appling
>Dept. of Chemistry & Biochemistry
>Univ. of Texas at Austin
>Austin, TX 78712
>(512)471-5842
>(512)471-5849 (FAX)
>_______________________________________________________________________________

Hi Dean

No problem.  
The method below is for nick-translated DNA but works for random priming,
kinasing oligos etc etc.  Only the Rf of the unincorporated label will
vary.  The Rf of the DNA is 0 (i.e. the origin) and free Pi is 1 (i.e.
solvent front).  dCTP, etc all vary between 0.5 & 0.9 which will become
obvious to you on autoradiography

.c.PEI-CELLULOSE THIN LAYER CHROMATOGRAPHY
During pre-incubation, a sample (approx. 0.1  l, although neither the
volume nor its accuracy is important) is removed and spotted onto an origin
mark on a 4 x 8 cm (approx.) sheet of PEI-cellulose
(polyethyleneimine-cellulose coated plastic sheets for thin layer
chromatography; Merck #5579).  On completion of the labelling reaction,
a second small sample is removed and spotted adjacent to the first.  The
samples are air-dried, chromatographed for 10-15 min (or until the solvent
front approaches the top) in a foil-covered beaker containing approx. 2 mm
(depth) of 0.75 M KH2PO4 adjusted to pH 3.5 with orthophosphoric acid, then
wrapped in plastic film and autoradiographed for 10-30 min:
 
Labelling efficiency can be estimated by visual inspection of the
autoradiograph or by scanning the chromatogram with a hand-held radiation
monitor.  If greater than 95% of the label remains at the origin (i.e. in
the DNA) the labelling has proceded correctly.  If desired, a quantitative
measure of the relative levels of radioactivity in dCMP, dCTP, and DNA can
be obtained by scintillation or Cerenkov counting of appropriate areas
scraped from the chromatogram, using the autoradiograph as a template.

Good Luck, Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"If you don't do: you rust"
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