Dynabeads™ DNA DIRECT™ Universal Kit

High PCR background could be caused by too high a concentration of template DNA, primers, Mg2+, or dNTPs. To fix this, reduce the amount of template DNA, primers, enzyme, and Mg2+ used, elute the DNA prior to PCR amplification, and/or try performing hot-start PCR. High background can also be caused by contamination. Ensure that the supernatant is completely removed at each washing step to avoid carryover.

We recommend eluting the DNA in Resuspension Buffer, water, or low ionic strength buffer by incubation at 65 degrees C for > 5 mins. The complex must be fully resuspended before elution. The elution buffer can be pre-heated to 65 degrees C. After incubation, place tube in magnetic stand for 30 secs and transfer supernatant to a clean tube. To determine the elution efficiency, you can run some of the eluted DNA on an agarose gel. Alternatively, you can perform a PCR with the Dynabeads magnetic beads to determine if there is residual DNA remaining on the beads. Using more pre-heated elution buffer would be helpful to improve the elution efficiency.

PCR may be inhibited by inhibitors, or an insufficient number of PCR cycles might have been used. For inhibitors, ensure that the washing buffer is brought to room temperature for use, and add the buffer more vigorously. You can also ensure that the supernatant is completely removed at each washing step, or introduce an additional washing step.

Please review the following possibilities for why your Dynabeads magnetic beads are not pelleting:

- The solution is too viscous.
- The beads have formed aggregates because of protein-protein interaction.

Try these suggestions: - Increase separation time (leave tub on magnet for 2-5 minutes)
- Add DNase I to the lysate (~0.01 mg/mL)
- Increase the Tween 20 concentration to ~0.05% of the binding and/or washing buffer.
- Add up to 20 mM beta-merecaptoethanol to the binding and/or wash buffers.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

For biotin-labeled DNA that is less than 1 kb, we recommend you use Dynabeads M270 Streptavidin (Cat. No. 65305) and MyOne C1 magnetic beads (Cat. No. 65001). We recommend our Dynabeads KilobaseBINDER Kit (Cat. No. 60101), which is designed to immobilize long (>1 kb) double-stranded DNA molecules. The KilobaseBINDER reagent consists of M-280 Streptavidin-coupled Dynabeads magnetic beads along with a patented immobilization activator in the binding solution to bind to long, biotinylated DNA molecules for isolation. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html) for more information in regards to long biotinylated DNA fragment isolation.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.