Peaks from 70 – 90 bp (see the trace below) are primer dimers from the 2nd PCR that result from incomplete removal of small molecular materials during the final magnetic bead purification. These are usually caused by inaccurate pipetting of magnetic beads volume, poor mixing prior after adding magnetic beads, and/or incomplete removal of supernatant …
If the library yield is low, but other wise clean and free from by-product peak(s), the library may require optimization in 2nd PCR cycles. Increase or decrease the 2nd PCR cycles by 2 to 3. If significant by-product peak is present, please refer to FAQ’s above for detailed troubleshooting suggestions.
CleanPlex and CleanPlex UMI workflow generates a single peak of library of approximately 8,000 – 20,000 pM when measured with a fragment analyzer such as Agilent™ 2100 Bioanalyzer instrument and Agilent™ high sensitivity DNA reagents, or approximately 1.5 – 4 ng/µl when measured by Qubit™ dsDNA HS Assay Kit, depending on each specific panel. It …
What is the typical yield of a CleanPlex or CleanPlex UMI library? Read More »
For a CleanPlex panel with two or more primer pools, follow the procedure shown below. For CleanPlex UMI panels, the starting mPCR reaction is 40 uL, but is combined in a likewise fashion for total of 80 uL volume for downstream steps.
Possibilities include: Using incompatible index primers. DNA quantification was inaccurate, especially if using spectrophotometric methods such as nanodrop, OR DNA quality is extremely poor. 30% ethanol instead of 70% ethanol OR using TE/H2O instead of 70% ethanol for DNA purification. Forgetting to add magnetic beads for any of the purification steps. Forgetting to add one …
There is little or no library peak when assayed with a fragment analyzer. Read More »
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Yes, please contact [email protected] for a quote.
We are yet to reach our limit of multiplexibility in our current portfolio of a wide range of panels. For standard CleanPlex technology, we have designed and delivered panels of up to 20,000 amplicons (approx. 400-genes) with great performance. Panels with higher amplicon targets may require more primer pools, which in tern require more DNA. …
Please see user guide for examples of each ready-to-use panels. When using a fragment analyzer such as Agilent Bioanalyzer 2100, the library peak(s) should fall between 200-400bp, depending on the specific panel. A library can exhibit a single peak (such as OncoZoom) or multiple peaks (such as TP53) depending on the panel’s amplicon length distribution. …
What should I expect my NGS library to look like on a fragment analyzer trace? Read More »
Support documents In addition to the User Guide, please download and review the Quick Guide from the Product Document page for details on some often overlooked but critical steps. You can also refer to the Videos page for the series of short videos that demonstrate the Cleanplex protocol step by step. DNA Quality and Quantification The quality …