CleanDTR Instructions for Use V.5 - FEBRUARY 2025 CDTR-0005, CDTR-0050, CDTR-0500 CleanNA, Coenecoop 75, 2741 PH, Waddinxveen, The Netherlands

Information in this document is subject to change without notice. Disclaimer For Research Use Only. Not for use in diagnostic procedures. CleanNA disclaims all warranties with respect to this document, expressed or implied, including but not limited to those of merchantability or fitness for a particular purpose. To the fullest extend allowed by law, in no event shall CleanNA be liable, whether contract, tort, warranty, or under any statute or on any other basis for special, incidental, indirect, punitive, multiple or consequental damages in connection with or arising from this document, including,...

Introduction and Principle CleanDTR is an efficient paramagnetic bead-based system, designed to remove unincorporated dye terminators from Sanger sequencing reaction. The CleanDTR process involves three simple steps including bind, wash and elute. While binding the sequencing product selectively to the magnetic particles, unincorporated dyes, nucleotides, salts and primers will be removed during ethanol washes. This principle allows for elution of the pure Sanger Sequencing product in the elution buffer of choice. The protocol can be adapted to your current liquid handling workstation (e.g. CleanXtract...

Do not use CleanDTR after the expiration date on the label. Read the instructions carefully before using the kit. Make sure that the kit bottle is not damaged and that no liquid leaked from it. Do not use a kit that has been damaged.

When working with chemicals, always follow your facility's procedures and universal precautions by using disposable gloves, safety glasses, a labcoat etc. For all safety information, please consult the safety data sheet (SDS). Request your SDS via www.cleanna.com/resource-request. Note: For safe disposal, please consult your local waste regulations. CleanNA produces each lot of CleanDTR according to predetermined and validated protocols in the Quality Management System (QMS). Additionally, a quality check after production of each lot is performed to secure consistent product quality. CleanNA's...

Materials and reagents to be supplied by user for CleanDTR protocols: • 96- or 384-well PCR plate containing sequencing samples • Magnetic separation device, recommended for 96 samples is the Clean Magnet Plate 96-Well RN50 (Part# CMAG-96-RN50) • Multichannel pipettor • Multichannel Disposable Reservoirs • 85% ethanol (freshly prepared from non-denatured alcohol) • Elution Buffer (0,1 mM EDTA pH 8.0, molecular biology grade water or formamide)

• Thoroughly resuspend the magnetic beads by vortexing. 1. Vortex or shake CleanDTR vigorously to fully resuspend the magnetic particles. 2. Add 10 pL of CleanDTR to each well. /j\ Note: Add 10 pL of CleanDTR regardless of the volume of the sequencing reaction. 3. Add freshly prepared 85% ethanol according to the table below and mix thoroughly by pipetting or vortexing. /j\ Note: 85% ethanol must be freshly made. 4. Incubate at room temperature for 2-5 minutes. 5. Place the plate on a magnetic separation device for 2-5 minutes or until the particles are completely cleared from solution. 6. Aspirate...

9. Leave the plate on the magnetic separation device and let dry at room temperature for up to 5 minutes, or until the bead pellet is visibly dry but the surface still glossy. A\ Note: Do not over-dry the beads. Do not dry at higher temperature or under vacuum. 10. Remove the plate from the magnetic separation device. Resuspend dried beads with 40 pL of PCR-grade water, 0.1mM EDTA (pH 8.0), or formamide. Mix thoroughly by piptetting or vortexing. Ensure beads are no longer attached to the side of the well. 11. Incubate the resuspended beads at room temperature for 2-5 minutes. 12. Place the plate...

• Thoroughly resuspend the magnetic beads by vortexing. 1. Vortex or shake CleanDTR vigorously to fully resuspend the magnetic particles. 2. Add 5 pL of CleanDTR to each reaction of a 384-well PCR plate. /!\ Note: Add 5 pL of CleanDTR regardless of the volume of the sequencing reaction. 3. Add freshly prepared 85% ethanol according to the table below and mix thoroughly by pipetting or vortexing. /j\ Note: 85% ethanol must be freshly made. 4. Incubate at room temperature for 2-5 minutes. 5. Place the plate on a magnetic separation device for 2-5 minutes or until the particles are completely cleared...

11. Incubate the resuspended beads at room temperature for 2-5 minutes. 12. Place the plate on the magnetic separation device for 2-5 minutes or until the sample appears clear. 13. Transfer the clear sample to a new plate. The samples are ready for loading on the sequencer. Alternatively, the samples can be stored at 4°C for up to 24 hours, or at -20°C for up to a month.

Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact your local distributor.

Ordering Information Contact your local distributor to order.

Manual Version

Contact Coenecoop 75 | 2741 PH Waddinxveen | The Netherlands +31 (0) 182 22 33 50 | [email protected] www.cleanna.com