Checking labelling oligo
m.rattray at umds.ac.uk
Wed Feb 19 08:29:37 EST 1997
> Is there is any simple method to check labelling incorporated to
> oligomere, if the oligonucleotide labelled with FITC-11-dutp. If you
> have any idea please contact me.
There is a very simple method which works well for us, it is based on a method that we got from
1. Cut two pieces of positively charged nylon membrane (e.g.Hybond N+)
2. Spot 1 ul of a series of dilution of your FITCdUTP, (1/10, 1/40, 1/120, 1/360, 1/1080, 1/3240)
onto each piece of membrane
3. Also spot 1ul of your probe onto each membrane
4. Leave the membranes for a few minutes till the spots dry a little
5. Wash one membrane at 60oC for 15-20 minutes in 2xSSC, 0.1% SDS.
6. Rinse both membranes with a little water (<10 seconds)
7. Pour a little 100% ETHANOL over both membranes
8. Wrap membranes in clingfilm and view on an ultraviolet light transilluminator.
The SSC+SDS washed membrane spots will retain fluorescence only if fluorescein has been
incorporated into the probe. The unwashed filter spots will retain all their fluorescence, so can
be used as a comparison of how well the probe labelling has worked: it is possible to ESTIMATE
the amount of fluorescein has been incorporated into the probe. (Unwashed filters with
fluorescein-UTP spots can be stored for several months in the dark at -20 oC)
We mainly use riboprobes, and good probes give a fluorescence similar to that of the 1/360 spot.
We throw away probes that don't meet this criteria - it saves us the time and trouble of trying
to use a bad probe.
I hope this works for you
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Molecular Neuropharmacology Laboratory
UMDS Division of Biochemistry & Molecular Biology
Tel: +44 (0)171 955 2831 Web: http://www.umds.ac.uk/neupharm/
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