Check the purity of the water you use I had some issues with the kit like what you are describing. Our ro/di water system was not purifying well and therefor the the transferase never fell off the dna due to the ions in the water therefor a shifted band appeared in many samples. When i switched to different water source the problem went away.

I had this kind of  problems with this kit. I solved it by doing ethanol precipitation after labelling. Now I get beautiful results. Good luck!

Can you send me your protocol of ethanol precipitation? Lizette

When I run ss and annealed oligos on PAGE they were fine.

The protocol I use is the following:

After DIG labelling you have 10 ul reaction, so you add 2ul of 4M LiCl abd 60ul of ice-cold absoulte ethanol, then you incubate for 2 hour at -70°C.  Centrifuge for 15 mins at 13000 xg, then wash with 100 ul of 70% ice-cold ethanol and recentrifugate fo an additional 15 mins.

Dry very well the pellet and resuspend in 10ul of TEN buffer. Now you can check the labelling as usual. 

Thanks a lot. Did you purify it after labelling? Does it effect labelling. Reaction volume after labelling is 25ul.

Yes, I purify it after labelling. It does not affect labelling at all, but still check its efficiency after ethanol precipitation. 

If you have 25 ul just add 5 ul of of 4M LiCl and 150ul of ice-cold absoulte ethanol, then continue with the rest of the protocol as it is written above. Good luck, hope it helps you!!!

Thanks a lot. I'll try this protocol and hope it will work.

I guess it is probe complexes because of the anealing. You probably need to run the probe on PAGE and cut out the ideal band and recycle it.