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

Robert_SB

Device: Fragment Analyzer 5300, 96 capilar array (33 cm)
Kits: Agilent DNF-915 Reagent Kit (35 – 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 µL/well with a 20 µL 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

Parents
  • 0

    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". 

Reply
  • 0

    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". 

Children
  • 0 in reply to Ian.J

    Hi Ian,
    Thanks for the quick response.

    I am using the EppendorfTm twin.tecTm PCR Plates, which are recommended in the manual.

    The injections for both marker and sample plate for both DNA kits were done at 5 kV for 5 s, which I am assuming is the correct method, since it was the first time I used it.

    The ladder question is interesting, though. I was already wondering about the meaning of "ready-to-use ladder". The quick guides of both kits state that 2 ul of ladder (or sample) has to be added to 22 ul 1x TE buffer. But if "ready-to-use" implies that 24 ul should be added to the well without extra 1x TE buffer this might explain the really low intensities (at least of the ladder).

    Okay. I just checked the Quick Guides on the Agilent website, which are updated versions to the ones the technician put on our desktop PC for reference three months ago Disappointed
    The latter are clearly outdated, describing the method mentioned above (2 ul ladder to 22 ul 1xTE). The new versions more or less clearly state that 24 ul of ladder are added to the well.

    Hence, case solved.

    Thanks for the assistance.


    Best regards,
    Robert

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