probe: incorporation measurement

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Wed Mar 30 02:38:55 EST 1994


In response to a number of requests I am posting the following with regard
to measuring the amount of incorporation of label into probes.  I have a
picture of the scan but it doesn't want to transfer to send.


The method for nicktranslation is given but it works with oligos as well.

PEI-CELLULOSE THIN LAYER CHROMATOGRAPHY
During pre-incubation, a sample (approx. 0.1 ul, 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 by "listening" at the origin using a lead shield with a small slit.
 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.
Trouble-shooting guide
Problems are readily diagnosed from autoradiography of PEI-cellulose
chromatograms:

dCTP runs at about 1/2 RF and dCMP higher

Excess dCTP     Excess dCMP     Solution  
        +       -       Increase time/concentration of DNase I pre-incubation
        +       +       Increase time/concentration of DNase I pre-incubation
        -       +       Decrease time/concentration of Pol I incubation

PI travels with the solvent front.  
This method is very good for monitoring endlabelling which I do for 15 min
at 37 only with better than 95% incorporation.  After that the Pi comes off
again and can be easily be seen on the autorad.


Cheers and Good Luck, Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
PO Box 334, Canberra, ACT 2600, Australia
E-mail: Klaus.Matthaei at anu.edu.au

"I'd rather a bottle in front of me than a frontal lobotomy"
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