Home Resources Video Library NEBNext® Tech Tips - Bead drying of cleanup steps after APOBEC

NEBNext® Tech Tips: Bead drying of cleanup steps after APOBEC

This video includes tips for handling the unique bead-drying properties seen during NEBNext EM-seq™ and NEBNext E5hmC-seq™ workflows.

Script

All EM-seq and E5hmC-seq workflows use APOBEC for deamination of DNA. The post-APOBEC cleanup occurs at different stages, depending on the workflow, either immediately after APOBEC treatment or following PCR. In either case, sample purification beads behave differently than they do in standard library prep cleanups. Following the APOBEC step, the beads dry very quickly and must be eluted before they become too dry, or the final library yield will be significantly reduced. Therefore, it is important to watch for over-drying at this step. 
Note that the time it takes to properly dry the beads depends on the humidity and conditions of your working environment. Following the ethanol wash when obvious droplets of ethanol have evaporated, the beads may still appear glossy, but at this stage it is okay to elute the DNA. Overdried beads appear matte, lighter brown and the pellets may appear cracked. In order to prevent over -drying your beads, work in small batches. Samples dried on beads will exhibit reduced recovery. Once beads have been added to the samples, it might be easier to stagger the clean up steps to avoid over drying the beads. It is okay to leave the sample with the beads added, or in 80% ethanol for more than 30 seconds while finishing supernatant removal and addition of the next buffer to the first set of samples. 
Remove the supernatant or ethanol only from as many samples as can be comfortably done before the beads dry out. Add the next ethanol wash or elution buffer to these first samples before the beads dry out. Then remove the supernatant or ethanol from the next set of samples. To enable processing of an entire experiment’s samples, its ok to leave samples in ethanol for longer than 30 seconds. Beads eluted at the correct time will resuspended with gentle up and down pipetting. 
Beads that are overly dried will remain in clumps and be difficult to resuspend, which will affect sample recovery. Thanks for watching. If you have any additional questions, please reach out to us at info@neb.com.
 

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