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 and quantity of the starting input DNA is critical in determining the appropriate cycles numbers and cycling conditions in the following steps of the workflow. We recommend using a fluorometric method such as the Qubit High Sensitivity dsDNA kit to determine the concentration of your starting materials. Do not use a spectrometric method such as the Nanodrop because it can significantly overestimate your starting concentration. High quality sample and higher quantity input generally yield higher quality libraries.
Correct Cycling Conditions
Generally, a thermal cycler set with the highest ramp speed, such as 5°C/second and higher, is not recommended. For thermal cyclers with adjustable ramp speed, we recommend 3°C/second up and 2°C/second down speed, or use the default setting (no ramp adjustment).
Making Working Solutions & Vortexing Well
Ensure accurate assembly of working solutions by pipetting viscous reagents carefully. Remove excess liquid from outside of the pipette tip and pipette mix as needed.
Mixing combined solutions is also CRITICAL. Vortex or pipette mix as needed to ensure all PCR working solutions, Digestion working solutions, and all bead+ sample solutions are homogeneous prior to starting the thermocycling or incubations.
Accurate Bead Dispense
The magnetic bead to sample volume ratio is critical to a clean library. Remove excess liquids from outside of tips and pipette mix as needed. Mix combined solution THOROUGHLY prior to incubation.
Cleanplex protocol is designed as a single tube workflow. With the exception of combining multi-pool panel reactions after the multiplex PCR step, solutions do not require tube to tube transfer, thus avoiding loss of sample and volume. Ensure the magnetic rack used is intended for the PCR tube or PCR plate being used. Avoid disturbing and removing beads during any of the washing steps.
Thorough Ethanol Washes
Ensure the cleanest libraries by carefully removing all droplets from supernatant and ethanol washes. This minimizes carry over of byproducts that would be amplified in the final PCR step.