Fragment Analyzer 5300 - Wrong peak intensities when using DNA kits.

Question

Device: Fragment Analyzer 5300, 96 capilar array (33 cm) Kits: Agilent DNF-915 Reagent Kit (35 &ndash; 5000 bp), Agilent DNF-905 dsDNA Kit (1-500 bp) 
 Hi, I have the issue that peak intensities of the lower and upper marker are super high whenever I use DNA kits (so far I tested the two DNA kits mentioned above). Due to the high intensities, samples and ladders are almost non-existent (images of ladder attached below). I don't have the issue when running RNA kits, though. Here the peaks are normal and they are looking exactly as described in the manual. 
 What could be possible reasons for this? 
 I prepared the Marker Plate as described in the manual: "When preparing lower/upper DNA marker plates for repeated use, a volume of 30 &micro;L/well with a 20 &micro;L mineral oil overlay is recommended." 
 Many thanks, Robert 
 
 100 bp ladder on DNF-915 
 
 100 bp ladder on DNF-905 
 
 35-400 ladder on DNF-905 
 
 RNA ladder on DNF-471

Ian.J · Answer

Hello Robert, 
 While I would consider the marker a little higher than expected, the ladder intensity for DNF-905 and DNF-915 are significantly lower than expected. I would suggest reviewing the possible contributing factors below. 
 
 DNF-905 and DNF-915 kits are qualitative kits with two injections, one from the marker plate and one from the sample/ladder plate.
 
 Ensure compatible plates are being used for both the sample/ladder plate and the marker plates. 
 Review the method for possible changes to the injection parameters of the sample injection.

The ladders for DNF-905 and DNF-915 come ready-to-use.
 
 Ensure the ladder is not being diluted in Marker solution.

If the problem persists, please reach out to your local Agilent Technical Support team. When contacting technical support, it would be beneficial to share the data files in question. To export the data files, please open them in ProSize and use the menu Help>"Zip Opened Data file".