DNA high sensitivity assay, strange ladder and marker

Dear Agilent team,

I've run this assay many times, but today I got a result I've no clue how to explain or where to begin troubleshooting.

The gel-dye mix is about 1.5 months; previous experience shows no major impact on the result. 

The ladder and other reagents were mixed well and stood at room temperature for over 30 minutes. 

Here's the result of ladder, and one of the samples (sheared DNA). 

(1) Ladder showed weird escalating baseline, 

(2) sample showed a steady baseline but missing lower and upper markers. 

I appreciate any suggestions or solutions.

Thank you in advance.

  • Dear SYChen,

    Thank you for contacting the Agilent team. To address the issue related to sub-optimal ladder profile and missing markers in sample well, I would do the following:

    1. Maintenance of the pin-set as described on page 128 of the M&T guide here and execute the short circuit test after ensuring that the pin-set is clean and dry. Additionally, here is a video that describes the process. This is suggested because a contaminated pin-set can affect optimal migration and can lead to raised baseline.
    2. Maintenance of the priming station following steps as outlined starting page 134 of the M&T guide above and as described in our Agilent Knowledge Portal article here .
    3. Please make sure that prepared gel-dye mix is used with 6 weeks of preparation. Additionally, syringe clip setting should be at the lowest position for DNA high sensitivity assay. Please refer to the kit guide for details.
    4. Test run. After performing above steps, please consider doing a test run without samples. For the test run, I would use an unexpired kit and prepare the chip exactly as you would when running samples but instead of samples put 1 ul of TE buffer. Before the test run:
      • Please ensure that the kit/reagents are not expired and stored correctly at 4C.
      • Allow reagents to warm up to room temperature for 30 minutes prior to preparing the chip.
      • Please prepare fresh gel-dye mix for the test run

    If test run per step 4, looks ok then it is ok to proceed with running your own samples. However, please make sure that samples are loaded within the sizing, concentration range of the kit, and sample buffer is compatible with DNA high sensitivity kit per limits published in kit guide.

    If further assistance is needed, then please share the raw data files ending with .xad with your local support at service@welgene.com.tw . Default location for .xad files is C:>Program Files (x86)>Agilent>2100Bioanalyzer>2100 expert>Data.

    Thank you, Shweta

  • Thank you so much for the quick response and detailed procedure for solving the issue! I think I'll contact local support for 1 and 2, and followed 4 to do the test run.



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