Checking labelling oligo

[Marcus Rattray]

Balwi wrote:
> 
> 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.
> 
> Balwi

There is a very simple method which works well for us, it is based on a method that we got from 
Amersham.  

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


Marcus Rattray


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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/