There are peaks from 70 – 90 bp in addition to the main library peak on the Agilent™ 2100 Bioanalyzer chart?
There is a peak around 150 bp in addition to the main library peak on the Agilent™ 2100 Bioanalyzer chart.
Significant peaks from 150 – 160 bp may be caused by under-amplification of the library (see the graph below on the right). Certain panels may tend to produce more non-specific amplification products. In this case, two rounds of magnetic bead purification after the digestion step solves the problem.
(1) 30% ethanol instead of 70% ethanol is used in DNA purification with magnetic beads; or
(2) forgetting to add magnetic beads after digestion. The workflow does not require users to change tubes, nor to remove magnetic beads from previous steps. One can easily tell if magnetic beads in each purification steps are added or not by checking the volume; or
(3) forgetting to add Stop Buffer after digestion, especially when handling a large number of samples.
(4) over-drying beads can decrease library yield, whereas under-drying beads can leave ethanol that will inhibit PCR.
(5) inaccurate volume of magnetic beads added. Ensure magnetic bead aliquot is well mixed prior to each dispense.
(6) insufficient mixing. Ensure all reactions and solutions are thoroughly mixed and homogeneous prior to proceeding to thermocycling and incubation steps.
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