DNA fragments observed in agarose gel are not visible in capillary electrophoresis

Hello, as a laboratory, we are a 12-row 5200 Fragment Analyser user. We are having problems getting results for our SSR-based experimental studies. When we load our samples to the device after PCR (5ul PCR sample + 19ul Dilution Buffer), we expect to see a band profile of approximately 200-300bp. However, the results we get are as if the reaction never worked. Then, when we load the same sample in the same volume (5ul) on agarose gel at 1.5% concentration, we can see DNA fragments of the size we expect. So the PCR is working.

To understand the problem, we ran another set of samples that were tested before the samples mentioned. The results were very good, even though the samples were run a second time and had been in the fridge for more than 2 months. After this run, we again loaded the problematic samples in a different volume (10ul sample + 14 Dilution Buffer) and the result was again negative. Different people performed the PCR and capillary loading steps, but the result was still unchanged.

We are waiting for your suggestions and support.

Here two photos of agarose jel and capillary results.

  • Hello,

    Based on the ladder profile in the screenshot you shared, it appears that you are running the DNF-905 kit.

    In reference to the issue of missing sample signal or blank lanes, since ladder and marker peaks are present as expected, issue is likely sample-related specifically. Therefore, for optimal assay performance, it is recommended to ensure that samples are loaded per specifications indicated in the kit guide. For example, you indicated that sample was prepared by adding 5ul of sample + 19ul of dilution buffer, but for the DNF-905 kit sample, it is recommended to prepare using 2uL of sample and 22uL of dilution buffer. Changing this ratio can change sample buffer concentration which can affect proper electrophoresis of the samples.

    Further, blank lanes can be due to missed injections due to incompatible (too tall) plasticware. Please refer to page 141 of the Fragment Analyzer System Manual for approved plastics. 

    If there are still issues, please email the zipped data file to your local support team from here: Contact Us | Agilent

    The zipped data file can eb prepared by opening the data in ProSize, then going to Help > Zip Opened Data File.

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